Mutant protein and refolding method

ABSTRACT

An FrpB protein having one or more deletions of non-conserved amino acids compared to a corresponding wild-type FrpB protein, and a method of refolding an FrpB protein.

FIELD OF THE INVENTION

The present invention relates to mutants of FrpB—an outer membraneprotein of Neisseria meningitidis organisms, to a method of refoldingFrpB proteins, to such refolded proteins, pharmaceutical compositionscomprising FrpB proteins, and their use in the treatment, prevention anddiagnosis of bacterial infections, such as Neisserial infections, andparticularly, but not exclusively, Neisseria meningitidis and/orNeisseria gonorrhoeae.

BACKGROUND OF THE INVENTION

Neisserial strains of bacteria are the causative agents for a number ofhuman pathologies, against which there is a need for effective vaccinesto be developed. In particular Neisseria gonorrhoeae and Neisseriameningitidis cause pathologies which could be treated by vaccination.

Neisseria gonorrhoeae is the etiologic agent of gonorrhea, one of themost frequently reported sexually transmitted diseases in the world withan estimated annual incidence of 62 million cases (Gerbase et al 1998Lancet 351; (Suppl 3) 2-4). The clinical manifestations of gonorrheainclude inflammation of the mucus membranes of the urogenital tract,throat or rectum and neonatal eye infections. Ascending gonococcalinfections in women can lead to infertility, ectopic pregnancy, chronicpelvic inflammatory disease and tubo-ovarian abscess formation.Septicemia, arthritis, endocarditis and meningitis are associated withcomplicated gonorrhea.

The high number of gonococcal strains with resistance to antibioticscontributes to increased morbidity and complications associated withgonorrhea. An attractive alternative to treatment of gonorrhea withantibiotics would be its prevention using vaccination. No vaccinecurrently exists for N. gonorrhoeae infections. Neisseria meningitidis(meningococcus) is a Gram-negative bacterium frequently isolated fromthe human upper respiratory tract. It occasionally causes invasivebacterial diseases such as bacteremia and meningitis. Most caes ofdisease are in infants or young children. The incidence of meningococcaldisease shows geographical seasonal and annual differences (Schwartz,B., Moore, P. A., Broome, C. V.; Clin. Microbiol. Rev. 2 (Supplement),S18-S24, 1989). Most disease in temperate countries is due to strains ofserogroup B and varies in incidence from 1-10/100,000/year totalpopulation sometimes reaching higher values (Kaczmarski, E. B. (1997),Commun. Dis. Rep. Rev. 7: R55-9, 1995; Scholten, R. J. P. M., Bijlmer,H. A., Poolman, J. T. et al. Clin. Infect. Dis. 16: 237-246, 1993; Cruz,C., Pavez, G., Aguilar, E., et al. Epidemiol. Infect. 105: 119-126,1990).

Epidemics dominated by serogroup A meningococci, mostly in centralAfrica, are encountered, sometimes reaching levels up to1000/100.000/year (Schwartz, B., Moore, P. A., Broome, C. V. Clin.Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Nearly all cases as awhole of meningococcal disease are caused by serogroup A, B, C, W-135and Y meningococci and a tetravalent A, C, W-135, Y polysaccharidevaccine is available (Armand, J., Arminjon, F., Mynard, M. C., Lafaix,C., J. Biol. Stand. 10: 335-339, 1982).

The frequency of Neisseria meningitidis infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Neisseria meningitidis strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

Van der Ley et al (1996) Microbiology 142:3269-3274 reports that thereis at present no vaccine available against group B organisms, which arethe predominant cause of meningococcal disease. The use of the group Bcapsular polysaccharide as a vaccine has been hindered by the presenceof identical structures on neural cell adhesion molecules in the humanhost, raising the possibility of inducing autoimmune pathology with sucha vaccine. It has been proposed to use vaccines based on meningococcalouter membrane proteins. However, it has been found that a largeproportion of the protective antibodies induced are strain specific. Inaddition, not all outer membrane components contribute to the inductionof bactericidal antibodies. The FrpB protein from Neisseria is aniron-regulated outer membrane protein that shows the characteristics ofa TonB-dependent outer membrane receptor. It has a molecule weight ofaround 77 kDa; although this may vary between strains. It has beenthought that the FrpB protein may be a good candidate for the productionof bactericidal antibodies. However Ala'Aldeen et al (1994) Vaccine12:535 found that monoclonal antibodies (mAbs) isolated againstwild-type FrpB were found to be strain specific.

It has also been envisaged that recombinant FrpB expressed in cellscould be produced for use in such new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests. However, one of the majorlimitations on the expression of proteins is the inability of manyrecombinant proteins to fold into their biologically activeconformations. Often only low yields of the recombinant protein areobtained due to aggregation and mis-folding of the unfolded species.Indeed, protein refolding, in which the protein acquires it native andactive structure, is one of the biggest challenges in molecular biology.

Clearly, the need therefore remains to provide a vaccine againstNeisseria disease comprising purified protective outer membrane proteinsincluding mutant proteins in a refolded conformation suitable to elicitan effective immune response, preferably a cross-protective response.

SUMMARY OF THE INVENTION

The present invention provides a mutant FrpB proteins, more particularlyloop-deletion mutants, which may induce broad-spectrum bactericidalantibodies.

The present invention also provides a method for refolding the FrpBprotein, including both mutants and wild-type.

The present invention also relates to such refolded FrpB proteins.

The present invention also provides methods for using the proteins ofthe present invention, including their use in the prevention andtreatment of microbial diseases, amongst others, in e.g. subunit andouter membrane vesicle vaccines. In a further aspect, the inventionrelates to diagnostic assays for detecting diseases associated withmicrobial infections and conditions associated with such infections.

STATEMENTS OF THE INVENTION

According to one aspect of the present invention there is provided anFrpB protein having one or more deletions of non-conserved amino acidscompared to a corresponding wild-type FrpB protein. These proteins ofthe present invention are referred to generally as “mutant” proteins.The term “deletion” as used herein includes the substitution,replacement or mutation of one or more of the non-conserved amino acids.The substituted sequence may or may not be the same size as the sequenceit replaces. By “non-conserved” amino acids we include amino acidspresent in a wild-type FrpB protein from a first strain, but which maynot be present in a wild-type FrpB protein from a second strain. Theproteins of the present invention are isolated proteins. By “isolated”we include proteins that have been removed from their natural state orotherwise subjected to human intervention. Preferably the protein hasalso been purified to a level of more than 40, 50, 60, 70, 80, 90, 95 or99%.

The present invention is therefore broadly directed to proteins havingconserved amino acids of FrpB proteins.

Preferably the FrpB protein is one in which one or more of the aminoacids of at least one of its loops has been deleted.

Preferably the mutant protein according to this invention isnon-strain-specific, cross-reactive, and/or cross-protective. By“strain-specific” we include the generation of an immune response whichis directed to, or at least predominantly directed to, an autologous(homologous) N. meningitidis strain. By “cross-reactive” we include thegeneration of an immune response directed to one or more heterologousNeisserial, preferably N. meningitidis, strains. By “cross-protective”we include the generation of an immune response providing protectionagainst infection by one of more heterologous Neisserial, preferably N.meningitidis, strains. Preferably the protein of the present inventionis conformationally stable.

Preferably the protein is one in which one or more of the amino acids ofat least 2 loops have been deleted. The loops may be determined bysequence comparison. Preferably the protein is one in which one or moreof the amino acids of loop 7 and/or have been deleted. According to aparticular preferred embodiment the deletions are from loop 7. Inanother preferred embodiment the deletions are from loop 7 and loop 5.Loops 5 and 7 have been identified as being regions of FrpB proteinshaving relatively high frequencies of non-conserved amino acids. Thenumbering of the FrpB surface loops follows the numbering system used byPettersson et al (1995) Infect. Immunology 63:4181-4184 (FIG. 6);however corresponding deletions can be made according to other modelswhich are or become available (for instance that of FIG. 9, the “newmodel”, where loops 3 and 5 correspond roughly with loops 5 and 7,respectively, of the old model of FIG. 6). It will be appreciated thatcorresponding deletions may be determined using sequence comparisonmethods.

The protein may also have one or more amino acid deletions from any oneor more of loops 1, 2, 4, 6, 8, 9, 10, 11, 12 and 13 (or thecorresponding loops according to any other topological model [e.g. the“new model”]); although the present invention preferably comprises oneor more of these more constant regions of an FrpB protein.

In one embodiment the protein is one in which 11 to 33 amino acids havebeen deleted from loop 7, more preferably 23-33 amino acids, even morepreferably 28 amino acids have been deleted from loop 7.

In another embodiment the protein is one in which 18-29 amino acids havebeen deleted from loop 5, more preferably 19-29 amino acids, even morepreferably 24 amino acids have been deleted from loop 5.

In one embodiment there is provided a protein in which, with referenceto FrpB strain H44/76, the amino acid deletion is made in the range ofamino acids 376-413, more preferably 381-408, or a correspondingdeletion made, from loop 7.

In a preferred embodiment there is provided a protein in which, withreference to FrpB strain H44/76, an amino acid sequence comprisingTTEEKNGQKVDKPMEQQMKDRADEDTVH has been deleted, or a correspondingdeletion made, from loop 7.

In a preferred embodiment there is provided a protein in which, withreference to FrpB strain H44/76, the amino acid deletion is made in therange of amino acids 247-280, more preferably 252-275, or acorresponding deletion made, from loop 5.

In a further preferred embodiment there is provided a protein in which,with reference to FrpB strain H44/76, an amino acid sequence comprisingQHRGIRTVREEFTVGDKSSRINID has been deleted, or a corresponding deletionmade, from loop 5.

The present invention also provides a polynucleotide encoding the mutantprotein of the present invention.

According to another aspect of the present invention there is providedan expression vector comprising the polynucleotide of the presentinvention.

According to another aspect of the present invention there is provided ahost cell comprising the polynucleotide of the present invention.

According to another aspect of the present invention there is provided amethod for producing the mutant protein of the present inventioncomprising: culturing the host cell of the invention, and recovering theexpressed protein.

According to another aspect of the present invention there is provided amethod for refolding an FrpB protein (including both mutant andwild-type) comprising contacting the FrpB protein with an alkalinerefolding buffer comprising 3-dimethyldodecylammoniopropanesulfonate(Zwittergent 3-12 or SB-12).

In one embodiment the refolding buffer comprises ethanolamine and SB-12.

Preferably the ethanolamine is about 15-30, preferably about 20 mMethanolamine.

Preferably the refolding buffer has pH11.

Preferably the SB-12 is 0.2-1% SB-12 more preferably 0.3-0.8% SB-12,even more preferably 0.5% SB-12.

In a preferred embodiment the refolding buffer further comprises urea,NaCl (such as 0.4M NaCl) and/or guanidium chloride, more preferablyguandinium chloride, most preferably 0.4M guanidium chloride.

According to another aspect of the present invention there is provided amethod comprising (preferably all of) the following steps:

-   optionally expressing an FrpB protein in a host cell;-   optionally breaking the host cell to obtain an inclusion body    comprising the FrpB protein;-   optionally washing the inclusion body;-   optionally solubilisation of at least part of the inclusion body and    the FrpB protein, preferably with Guanidinium hydrochloride;-   contacting the solubilised FrpB protein with the refolding buffer;    and-   optionally removing the refolding buffer from the FrpB protein.

According to another aspect of the present invention there is providedrefolding buffer comprising ethanolamine, SB-12 and, optionally,guanidium chloride (in the concentrations mentioned above) for use inthe method of the present invention.

According to another aspect of the present invention there is providedan isolated, refolded FrpB protein (including a mutant protein) obtainedor obtainable by the method of the present invention.

According to another aspect of the present invention there is provided apharmaceutical composition comprising at least one FrpB protein of thepresent invention, and a pharmaceutically acceptable carrier.

Preferably at least 30%, 50%, 70%, or 90% of the FrpB protein present inthe composition is refolded.

Preferably the composition is in the form of a vaccine.

In one embodiment the pharmaceutical composition comprises a refoldedFrpB protein derived from Neisseria meningitidis.

In another embodiment the pharmaceutical composition comprises arefolded FrpB protein derived from Neisseria gonorrhoeae.

Preferably the pharmaceutical composition comprises at least one otherNeisserial antigen.

In one embodiment the pharmaceutical composition comprises at least oneother Neisserial antigen derived from Neisseria gonorrhoeae.

In another embodiment the pharmaceutical composition comprises at leastone other Neisserial antigen derived from Neisseria meningitidis.

In one embodiment the pharmaceutical composition is in the form of asubunit composition; although it may be admixed with an outer membranevesicle preparation.

In another embodiment the pharmaceutical composition is in the form ofan outer membrane vesicle preparation; although it may be mixed with asubunit composition.

Preferably said composition further comprises at least one Neisserialadhesin, or one other Neisserial outer membrane protein, or at least oneNeisserial autotransporter, or at least one Neisserial toxin, or atleast one Neisserial Fe acquisition protein. Most preferably, inaddition to isolated FrpB of the invention, at least one Neisserialautotransporter and at least one Neisserial toxin is present, or atleast one Neisserial autotransporter and at least one Neisserial Feacquisition protein is present, or at least one Neisserial toxin and atleast one Neisserial Fe acquisition protein is present, or at least oneNeisserial autotransporter and at least one Neisserial Fe acquisitionprotein and at least one Neisserial toxin is present, or immunogenicfragments thereof.

More preferably the pharmaceutical composition comprises at least onefurther antigen (or fragment thereof) selected from at least one of thefollowing classes:

-   at least one Neisserial adhesin selected from the group consisting    of FhaB, NspA Hsf, NadA, PilC, Hap, MafA, MafB, Omp26, NMB0315,    NMB0995 and NMB1119;-   at least one Neisserial autotransporter selected from the group    consisting of Hsf, Hap, IgA protease, AspA and NadA;-   at least one Neisserial toxin selected from the group consisting of    FrpA, FrpC, FrpA/C, VapD, NM-ADPRT, and either or both of LPS    immunotype L2 and LPS immunotype L3;-   at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA high, TbpA low, TbpB high, TbpB low, LbpA,    LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp,    NMB0964 and NMB0293; and-   at least one Neisserial membrane associated protein, preferably    outer membrane protein, selected from the group consisting of PldA,    NspA, TspA, FhaC, NspA, TbpA(high), TbpA(low), LbpA, HpuB, TdfH,    PorB, HimD, HisD, GNA1870, OstA, HlpA, MltA, NMB 1124,NMB 1162,NMB    1220,NMB 1313,NMB 1953,HtrA, TspB, PilQ and OMP85.

Most preferably the pharmaceutical composition further comprises atleast one other Neisserial antigen (or a fragment thereof) selected fromone or more of the following classes:

-   a) at least one Neisserial adhesin selected from the group    consisting of FhaB, Hsf and NadA;-   b) at least one Neisserial autotransporter selected from the group    consisting of Hsf, and Hap;-   c) at least one Neisserial toxin selected from the group consisting    of FrpA, FrpC, and either or both of LPS immunotype L2 and LPS    immunotype L3;-   d) at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA, TbpB, LbpA and LbpB; and-   e) at least one Neisserial outer membrane protein selected from the    group consisting of PldA, NspA, TspA, TspB, PilQ and OMP85.

Where the additional antigens are present on a bleb (OMV) they arepreferably upregulated in expression (compared to wild-type expressionlevels), for instance as described in WO 01/09350.

Preferably the pharmaceutical composition further comprises bacterialcapsular polysaccharides or oligosaccharides. The capsularpolysaccharides or oligosaccharides may be derived from bacteriaselected from the group consisting of Neisseria meningitidis serogroupA, C, Y, and/or W-135, Haemophilus influenzae b, Streptococcuspneumoniae, Group A Streptococci, Group B Streptococci, Staphylococcusaureus and Staphylococcus epidermidis, and are most preferablyconjugated to a source of T-helper epitopes.

According to another aspect of the present invention there is provideduse of an FrpB protein of the present invention (or a pharmaceuticalcomposition of the present invention) in the preparation of a medicamentfor use in generating an immune response in an animal.

Preferably the use is in the preparation of a medicament for treatmentof prevention of Neisserial infection. In one embodiment Neisseriameningitis infection is prevented or treated. In another embodimentNeisseria gonorrhoeae infection is prevented or treated.

According to another aspect of the present invention there is providedan antibody immunospecific for the FrpB protein of the presentinvention.

According to another aspect of the present invention there is provided apharmaceutical composition useful in treating humans with a Neisserialdisease comprising at least one antibody of the present invention and asuitable pharmaceutical carrier.

According to yet another aspect of the present invention there isprovided use of the antibody of the present invention (or compositioncomprising it) in the manufacture of a medicament for the treatment orprevention of Neisserial disease. In one embodiment Neisseriameningitidis infection is prevented or treated. In another embodimentNeisseria gonorrhoeae infection is prevented or treated.

According to a further aspect of the present invention there is provideda method of diagnosing a Neisserial infection, comprising identifying anFrpB protein, or an antibody as of the present invention, present withina biological sample from an animal suspected of having such aninfection, or by using an FrpB protein or antibody of the presentinvention to detect whether FrpB or antibodies against FrpB are presentwithin a biological sample from an animal. In one embodiment Neisseriameningitidis infection is diagnosed. In another embodiment Neisseriagonorrhoeae infection is diagnosed.

DETAILED DESCRIPTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example with reference to theaccompanying drawing in which:

FIG. 1 shows the amino acid and nucleotide sequences of FrpB strainH44/76;

FIG. 2 shows the amino acid and nucleotide sequences of FrpB strainFA19;

FIG. 3 shows the amino acid and nucleotide sequences of FrpB strain2996;

FIG. 4 shows the amino acid and nucleotide sequences of FrpB strain892257;

FIG. 5 shows sequence comparison of the mature FrpB protein fromNeisseria gonorrhoeae strain FA19 and Neisseria meningitidis strains892257, 2996, and H44/76;

FIG. 6 shows the topology of FrpB strain H44/76 in the outer membraneaccording to the model of Pettersson et al (1995) Infect. Immunology63:4181-4184;

FIG. 7 shows sequence comparison of loop 7 region FrpB from eightdifferent meningococcal strains. The H44/76 strain is used as thereference; and

FIG. 8 shows a gel demonstrating in vitro folding of urea-denaturedFrpB, FrpBΔ28 and FrpBΔ7Δ5.

FIG. 9 shows the new topology model of FrpB in its outer membrane, Loops3 and 5 (equivalent of loops 5 and 7, respectively, in the old methodare shown.

FIG. 10 shows A) acrylamide gel showing how FrpB is properly refolded,and B) CD spectrum recorded on a Jasco-810 spectropolarimeter [averageof 30 scans] showing that a highly folded product is obtained.

FIG. 11 Evaluation of the immuneresponse against in vitro folded FrpB(mutants) Western blot performed with the parent strain and 16heterologous meningococcal strains (first lane shows the parent strain).

FIG. 12: Dot blot analysis of in vitro folded FrpB-C1 and FrpB-C2. Invitro folded proteins were kept cold or heated to unfold the protein.Immunodetection was performed with mouse monoclonal antibody MN5C11G1:100,000 (α-PorA 1.7, 16).

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc(as well as the complete version Current Protocols in MolecularBiology).

The terms “comprising”, “comprise” and “comprises” herein is intended bythe inventors to be optionally substitutable with the terms “consistingof”, “consist of”, and “consists of”, respectively, in every instance.

FrpB Protein

FrpB is the most abundant iron-limitation-inducible outer-membraneprotein of Neisseria meningitidis. Thus, as used herein, the term “FrpB”includes Fe-regulated protein B and generally encompasses any proteinhaving an amino acid sequence identical, or substantially identical, tothe amino acid sequence of wild-type (also known as naturally occurring)FrpB. Further details on FrpB can be found in U.S. Pat. No. 6,265,567.“FrpB” and “FrpB protein” as used interchangeably herein. FrpB is alsobe known as FetA. As used herein the term “protein” includes the term“polypeptide”. The present invention relates to mutant forms of the FrpBprotein, as well as refolded FrpB protein, which refolded protein may ormay not be the mutant form. When the refolded protein is derived fromthe wild-type protein it is preferably an isolated, refolded FrpBprotein. By “isolated” we mean that the FrpB protein is free from otherproteins with which it is normal associated. The refolded FrpB proteinis preferably a recombinant protein. By “recombinant” we mean that theprotein has been obtained using the application of molecular biology.However the refolding method is also applicable to natural or syntheticproteins which require refolding, or purification by means of unfoldingand refolding. Preferably the isolated FrpB of the invention is purifiedin that it is more than 40, 50, 60, 70, 80, 90, 95, or 99% pure. Mostpreferably the FrpB of the invention is biologically pure in that it ismore than 40, 50, 60, 70, 80, 90, 95, or 99% free of other Neisserialproteins and/or other proteins of the host cell from which it was made.

The present invention provides mutant FrpB proteins. The term “mutant”in relation to the amino acid sequences of the present inventionincludes any substitution of, variation of, modification of, replacementof, deletion of or addition of one (or more) amino acids from or to theFrpB sequence providing the resultant amino acid sequence preferably iscapable of raising antibodies which recognise an FrpB protein (ifnecessary when coupled to a carrier). For ease of reference this isreferred to as deletion of one (or more) amino acids. The protein ismutated in the sense that it is varied from the corresponding wild-typeFrpB sequence, preferably such that the immune response is directed tonon-varied surface loops. Thus, the sequence may also be deleted in thesense that it is replaced by non-natural (a peptide from a differenceNeisserial protein, or from a completely different species, preferably aChlamydial peptide) sequence, preferably such that the immune responseis directed to non-varied surface loops. In one embodiment the sequenceis replaced with conserved sequence from another organism (e.g.Chlamydia trachomatis).

According to a preferred embodiment of the present invention at leastpart of one of the surface exposed loops of FrpB protein is deleted. Ina preferred embodiment, loop 7 and/or loop 5 is deleted, preferably loop7, more preferably a double mutant is generated. These preferreddeletions will now be described for ease of reference to Neisseriameningitidis strain H44/76. However corresponding deletions can be madein any Neisseria meningitidis strain, and indeed any Neisserial organismor strain, such as for example Neisseria gonorrhoeae. Such correspondingdeletions can be determined using sequence comparison as exemplified inFIGS. 5 and 7.

For ease we refer herein to the topological model of Pettersson;however, it will be appreciated that we could equally well refer to anyother model (e.g. the new model of FIG. 9, which will be specificallyreferred to if it is intended to be used in the context of thisinvention).

As mentioned above according to a preferred embodiment the mutantcomprises a deletion in loop 7. According to this embodiment (withreference to FrpB strain H44/76) in the range of 10 to 35 amino acidsare deleted from loop 7, preferably 15-33, or 20-33, and most preferably24 to 33 amino acids are deleted from loop 7. Preferably this deletionis made in the range of amino acids 376-413.

In a particularly preferred embodiment, about 28 amino acids are deletedfrom loop 7. This the amino acid deletion is preferably made in therange of amino acids 381-408. Thus preferably an amino acid sequencecomprising the sequence TTEEKNGQKVDKPMEQQMKDRADEDTVH is deleted fromloop 7.

With regard to deletions in respect of loop 5, (with reference to FrpBstrain H44/76) in the range of 7-30 amino acids are deleted from loop 5,preferably 10-29, or 15-29, and most preferably 19 to 29 amino acids aredeleted from loop 5. Preferably these deletions are made in the range ofamino acids 247-280.

In a particularly preferred embodiment about 24 amino acids are deletedfrom loop 5. Preferably this amino acid deletion is made in the range ofamino acids 252-275. Thus, preferably an amino acid sequence comprisingthe sequence QHRGIRTVREEFTVGDKSSRINID is deleted from loop 5.

As mentioned above corresponding deletions can easily be determined. Byway of example, such corresponding deletions will now be discussed inrelation to Neisseria gonorrhoeae strain FA19 and Neisseria meningitidisstrains 892257 and 2996. However the invention may be applicable to anyFrpB protein which is or becomes available.

With reference to FrpB strain FA19 preferably in the range of 11 to 21amino acids are deleted from loop 7. This amino acid deletion may bemade in the range of amino acids 381-406.

Preferably, with reference to FrpB strain FA19, about 16 amino acids aredeleted from loop 7. This amino acid deletion is preferably made in therange of amino acids 386-401. Thus, an amino acid sequence comprisingthe sequence TNEEKKKNRENEKIAK may be deleted from loop 7.

With reference to FrpB strain FA19 preferably in the range of 19 to 29amino acids are deleted from loop 5. This amino acid deletion ispreferably made in the range of amino acids 247-280.

Preferably with reference to FrpB strain FA19 about 24 amino acids aredeleted from loop 5. This amino acid deletion is preferably made in therange of amino acids 252-275. Thus, an amino acid sequence comprisingthe sequence QHRGIRTVREEFAVSEKNSRITIK may be deleted from loop 5.

Preferably, with reference to FrpB strain 2996, in the range of 18 to 28amino acids are deleted from loop 7. This amino acid deletion may bemade in the range of amino acids 358-390.

Preferably 23 amino acids are deleted from loop 7. This amino aciddeletion may be made in the range of amino acids 363-385. Thus, an aminoacid sequence comprising the sequence NGQDVAKPADQQAKDRKDEALVH may bedeleted from loop 7.

With reference to FrpB strain 2996 preferably in the range of 18 to 28amino acids are deleted from loop 5. This amino acid deletion may bemade in the range of amino acids 225-257.

Preferably about 23 amino acids are deleted from loop 5. This amino aciddeletion may be made in the range of amino acids 230-252. Thus, an aminoacid sequence comprising the sequence QHRGIRTVGEEFTVTNNSRLDLD may bedeleted from loop 5.

With reference to FrpB strain 892257 preferably in the range of 11 to 21amino acids are deleted from loop 7. This amino acid deletion is made inthe range of amino acids 360-385.

Preferably about 16 amino acids are deleted from loop 7. This amino aciddeletion may be made in the range of amino acids 365-380. Thus, an aminoacid sequence comprising the sequence TDEEKNKNRENEKIAK may be deletedfrom loop 7.

With reference to FrpB strain 892257 preferably in the range of 19 to 29amino acids are deleted from loop 5. This amino acid deletion may bemade in the range of amino acids 226-259.

Preferably 24 amino acids are deleted from loop 5. This amino aciddeletion may be made in the range of amino acids 231-259. Thus, an aminoacid sequence comprising the sequence QHRGIRTVREEFTVGAKDSRINIK may bedeleted from loop 5.

As indicated above at least some amino acids from at least one of thesurface loops of the FrpB protein (preferably loop 7 and/or loop 5) maybe replaced by non-natural, i.e. heterologous sequence. It will also beappreciated that shuffling of regions between FrpB proteins of differentNeisserial strains is possible. Preferably such replacement sequence isfrom another bacterial outer membrane protein. It is preferred that suchreplacement sequences are conserved, i.e. able to generate an immuneresponse against more than one strain of a bacterial organism. In oneembodiment the replacement sequence is also derived from Neisserialouter membrane proteins, such as Neisseria gonorrhoeae or Neisseriameningitidis. An example of such a suitable outer membrane protein isgiven in U.S. Pat. No. 5,912,336 which describes another Nessierial ironregulated protein, designated TbpA. Replacement sequence couldconveniently be derived from any one or more of loops 2, 3, 4, 5 and 8of TbpA. These loops correspond generally to amino acids 226-309;348-395; 438-471; 512-576 and 707-723 of TbpA respectively. Preferablyone or more of loops 4, 5 and 8 are incorporated.

Another example of such a suitable outer membrane protein is given inWO01/55182, which describes the NhhA (or Hsf) surface antigen fromNeisseria meningitidis. Replacement sequence could conveniently bederived from one or more constant regions of an NhhA protein generallydesignated as C1, C2, C3, C4 and C5. An example of another replacementsequence which could be used in the present invention is described in EP0 586 266.

Further Neisserial OMP loops that may be substituted for FrpB loopsparticularly loops 5 and/or 7) are PorA loop 4 [or variable region 2](see 20 http//neisseria.org/nm/typing/porA/); PorA loop 5 (described in“Topology of outer membrane porins in pathogenic Neisseria spp”, van derLey, Poolman, etc. . . . , Infect Immun 1991, 59, 2963-71; its sequencein PorA P1.7,16 (H44/76) loop 5 being: RHANVGRNAFELFLIGSGSDQAKGTDPLKNH);LbpA surface exposed loops 4, 5, 7, 10 and 12, corresponding to aminoacids 210-342, 366-441, 542-600, 726-766 and 844-871, respectively, with12 being preferred (sequence KGKNPDELAYLAGDQKRYSTKRASSSWST) [see Prinzet al. 1999 J Bacter. 181:4417 for further details on LbpA surface loopsincorporated by reference herein]; NspA surface exposed loops 1, 2, 3 or4, corresponding to amino acid sequence 25-54, 61-87, 103-129 and149-164, respectively, preferably where loop 2 (e.g.FAVDYTRYKNYKAPSTDFKLYSIGASA) and/or 3 (e.g.

ARLSLNRASVDLGGSDSFSQTSIGLGVL) is inserted (as these loops are quitesmall not all the FrpB loop 5 and/or 7 would be ideally removed tointroduce these loops, and if both are to be introduced, it is preferredthat they are introduced on loop 5 or 7 (or vice versa) in order to tryto preserve the conformational epitope that exists between loops 2 and 3of NspA) [see Vandeputte-Rutten et al 2003 JBC 278:24825 for moredetails on NspA loops, incorporated by reference herein]; any of thesurface exposed loops of Omp85 (see Science 2003 299:262-5, andsupporting online material Fig S4, incorporated by reference herein).

Alternatively peptide mimotopes of bacterial carbohydrate antigens maybe incorporated into FrpB in the above way. Preferably mimotopes ofNeisserial LOS are incorporated into loops 5 and/or 7 to advantageouslystimulate an immune response against this important antigen withouthaving its toxic effects in a vaccine. LOS mimotopes are well known inthe art (see WO 02/28888 and references cited therein, incorporated byreference herein).

It will be appreciated that the mutant proteins of the present inventionmay be prepared using conventional protein engineering techniques. Forexample, polynucleotides of the invention or coding for a wild-type FrpBmay be mutated using either random mutagenesis, for example usingtransposon mutagenesis, or site-directed mutagenesis.

A successful FrpB protein for the prevention of infection by Neisseriamay require more than one of the following elements: generation of serumand/or mucosal antibodies to facilitate complement mediated killing ofthe Neisseria, and/or to enhance phagocytosis and microbial killing byleukocytes such as polymorphonuclear leukocytes, and/or to preventattachment of the Neisseria to the host tissues; induction of a cellmediated immune response may also participate to protection.

The improvement of efficacy of a FrpB mutant of the invention can beevaluated by analyzing the induced immune response for serum and/ormucosal antibodies that have antiadherence, and/or opsonizingproperties, and/or bactericidal activity, as described by others(McChesney D et al, Infect. Immun. 36: 1006, 1982; Boslego J et al:Efficacy trial of a purified gonococcl pilus vaccine, in Program andAbstracts of the 24th Interscience Conference on Antimicrobial Agentsand Chemotherapy, Washington, American Society for Microbiology, 1984;Siegel M et al, J. Infect. Dis 145: 300, 1982; de la Pas, Microbiology,141 (Pt4): 913-20, 1995).

It will be understood that protein sequences of the invention or for usein the invention are provided as guidelines and the invention is notlimited to the particular sequences or fragments thereof given here butalso include homologous sequences obtained from any source, for examplerelated bacterial proteins, and synthetic peptides, as well as variants(particularly natural variants) or derivatives thereof. Loop sequencesgiven are meant as guidelines, and it is envisaged that any loopsequence comprising an epitope present in the loops described above maybe utilised.

Thus, the present invention encompasses variants, homologues orderivatives of the amino acid sequences of the present invention or foruse in the invention, as well as variants, homologues or derivatives ofthe amino acid sequences.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel. Although homology can also be considered in terms of similarity(i.e. amino acid residues having similar chemical properties/functions),in the context of the present invention it is preferred to expresshomology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Where a protein is specifically mentioned herein, it is preferably areference to a full-length protein but it may also encompass antigenicfragments thereof (particularly in the context of subunit vaccines).Preferred fragments include those which include an epitope. Particularlypreferred fragments include those with at least one surface loop. Withrespect to the mutants of the present invention this loop is preferablyother than loop 7 and/or loop 5. These fragments may contain or compriseat least 10 amino acids, preferably 20 amino acids, more preferably 30amino acids, more preferably 40 amino acids or most preferably 50 aminoacids, taken contiguously from the amino acid sequence of the protein.In addition, antigenic fragments denotes fragments that areimmunologically reactive with antibodies generated against theNeisserial proteins or with antibodies generated by infection of amammalian host with Neisseria. Antigenic fragments also includesfragments that when administered at an effective dose, elicit aprotective immune response against Neisserial infection, more preferablyit is protective against N. meningitidis and/or N. gonorrhoeaeinfection, most preferably it is protective against N. meningitidisserogroup B infection.

The present invention also includes variants of the proteins mentionedherein, that is proteins that vary from the referents by conservativeamino acid substitutions, whereby a residue is substituted by anotherwith like characteristics. Typical such substitutions are among Ala,Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp andGlu; among Asn and Gln; and among the basic residues Lys and Arg; oraromatic residues Phe and Tyr. Particularly preferred are variants inwhich several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted,deleted, or added in any combination.

The FrpB protein has also been identified in Neisseria gonorrhoeae (U.S.Pat. No. 6,265,567). The present invention may thus also be applied tothe FrpB protein from other Neisseria.

The FrpB produced by the present invention or mutant FrpB, fragment orvariant thereof, preferably is a product which displays at least some ofthe immunological activity of the wild type FrpB protein. Preferably itwill show at least one of the following:

-   An ability to induce the production of antibodies which recognise    the wild type FrpB (if necessary when the FrpB protein of the    present invention is coupled to a carrier);-   An ability to induce the production of antibodies that can protect    against experimental infection; and-   An ability to induce, when administered to an animal, the    development of an immunological response that can protect against    Neisserial infection such as Neisseria meningitidis or Neisseria    gonorrhoeae infection.

Preferably the mutant protein of the present invention is cross-reactiveand more preferably cross-protective.

The FrpB protein of the present invention is useful in prophylactic,therapeutic and diagnostic composition for preventing treating anddiagnosing diseases caused by Neisseria, particularly Neisseriameningitidis; although it may also have similar applications in relationto, e.g. Neisseria gonorrhoeae or Neisseria lactamica.

Standard immunological techniques may be employed with the FrpB proteinof the present invention in order to use it as an immunogen and as avaccine. In particular, any suitable host may be injected with apharmaceutically effective amount of the FrpB protein to generatemonoclonal or polyclonal anti-FrpB antibodies or to induce thedevelopment of a protective immunological response against a Neisseriadisease. Prior to administration, the FrpB protein may be formulated ina suitable vehicle, and thus we provide a pharmaceutical compositioncomprising a pharmaceutically effective amount of one or more proteinsof the present invention. As used herein “pharmaceutically effectiveamount” refers to an amount of FrpB (or other proteins of the invention)protein that elicits a sufficient titre of antibodies to treat orprevent infection. The pharmaceutical composition of the presentinvention may also comprise other antigens useful in treating orpreventing disease.

The FrpB protein of this invention may also form the basis of adiagnostic test for infection. For example, the present inventionprovides a method for detection of a Neisserial antigen in a biologicalsample containing or suspected of containing the Neisserial antigencomprising:

-   generating an anti-FrpB antibody using the protein of the present    invention;-   isolating the biological sample from a patient;-   incubating the anti-FrpB antibody or a fragment thereof with the    biological sample; and-   detecting bound antibody or bound fragment.

This invention also provides a method for the detection of antibodyspecific to FrpB protein in a biological sample containing or suspectedof containing said antibody comprising:

-   isolating the biological sample from a patient;-   incubating the FrpB protein of the invention with the biological    sample; and

detecting bound antigen.

This diagnostic test may take several forms including ELISA and aradioimmunoassay.

Further details on such applications are given below.

Bactericidal Assays

SBA Bactericidal Assays of the Invention

Cross-protection and an immunological response may be measured using aserum bactericidal assay (SBA) which is the most commonly agreedimmunological marker to estimate the efficacy of a meningococcal vaccine(Perkins et al. J Infect Dis. 1998, 177:683-691). For example animproved response of the mutant FrpB protein over the wild type proteinmay be characterised by the SBA elicited by the mutant FrpB being atleast 50%, two time, three times, preferably four times, five times, sixtimes, seven times, eight times, nine times and most preferably tentimes higher than the SBA elicited by the corresponding wild-typeprotein. Preferably SBA is measured against a homologous strain fromwhich the antigens are derived and preferably also against a panel ofheterologous strains. Thus, cross-protection may be measured if asatisfactory heterologous SBA is given against a panel of e.g. threeunrelated strains. Satisfactory SBA can be ascertained by any knownmethod. SBA can be carried out using sera obtained from animal models,or from human subjects.

A preferred method of conducting SBA with human sera is the following. Ablood sample is taken prior to the first vaccination, two months afterthe second vaccination and one month after the third vaccination (threevaccinations in one year being a typical human primary vaccinationschedule administered at, for instance, 0, 2 and 4 months, or 0, 1 and 6months). Such human primary vaccination schedules can be carried out oninfants under 1 year old (for instance at the same time as Hibvaccinations are carried out) or 2-4 year olds or adolescents may alsobe vaccinated to test SBA with such a primary vaccination schedule. Afurther blood sample may be taken 6 to 12 months after primaryvaccination and one month after a booster dose, if applicable.

SBA will be satisfactory for an antigen or bleb preparation withhomologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but preferably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthe strain of meningococcus from which the antigens of the inventionwere derived is greater than 30%, preferably greater than 40%, morepreferably greater than 50%, and most preferably greater than 60% of thesubjects.

Of course an antigen or bleb preparation with heterologous bactericidalactivity can also constitute bleb preparation with homologousbactericidal activity if it can also elicit satisfactory SBA against themeningococcal strain from which it is derived.

SBA will be satisfactory for an antigen or bleb preparation withheterologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but preferably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthree heterologous strains of meningococcus is greater than 20%,preferably greater than 30%, more preferably greater than 35%, and mostpreferably greater than 40% of the subjects. Such a test is a goodindication of whether the antigen or bleb preparation with heterologousbactericidal activity can induce cross-bactericidal antibodies againstvarious meningococcal strains. The three heterologous strains shouldpreferably have different electrophoretic type (ET)-complex ormultilocus sequence typing (MLST) pattern (see Maiden et al. PNAS USA1998, 95:3140-5) to each other and preferably to the strain from whichthe antigen or bleb preparation with heterologous bactericidal activityis made or derived. A skilled person will readily be able to determinethree strains with different ET-complex which reflect the geneticdiversity observed amongst meningococci, particularly amongstmeningococcus type B strains that are recognised as being the cause ofsignificant disease burden and/or that represent recognised MenBhyper-virulent lineages (see Maiden et al. supra). For instance threestrains that could be used are the following: BZ10 (B:2b:P1.2) belongingto the A-4 cluster; B16B6 (B:2a:P1.2) belonging to the ET-37 complex;and H44/76 (B:15:P1.7,16) belonging to the ET-5 complex, or any otherstrains belonging to the same ET/Cluster. Such strains may be used fortesting an antigen or bleb preparation with heterologous bactericidalactivity made or derived from, for instance, meningococcal strain CU385(B:4:P1.15) which belongs to the ET-5 complex. Another sample strainthat could be used is from the Lineage 3 epidemic clone (e.g. NZ124[B:4:P1.7,4]). Another ET-37 strain is NGP165 (B:2a:P1.2).

Processes for measuring SBA activity are known in the art. For instancea method that might be used is described in WO 99/09176. In generalterms, a culture of the strain to be tested is grown (preferably inconditions of iron depletion—by addition of an iron chelator such asEDDA to the growth medium) in the log phase of growth. This can besuspended in a medium with BSA (such as Hanks medium with 0.3% BSA) inorder to obtain a working cell suspension adjusted to approximately20000 CFU/ml. A series of reaction mixes can be made mixing a series oftwo-fold dilutions of sera to be tested (preferably heat-inactivated at56° C. for 30 min) [for example in a 50 μl/well volume] and the 20000CFU/ml meningococcal strain suspension to be tested [for example in a 25μl/well volume]. The reaction vials should be incubated (e.g. 37° C. for15 minutes) and shaken (e.g. at 210 rpm). The final reaction mixture[for example in a 100 μl volume] additionally contains a complementsource [such as 25% final volume of pretested baby rabbit serum], and isincubated as above [e.g. 37° C. for 60 min]. A sterile polystyreneU-bottom 96-well microtiter plate can be used for this assay. A aliquot[e.g. 10 μl] can be taken from each well using a multichannel pipette,and dropped onto Mueller-Hinton agar plates (preferably containing 1%Isovitalex and 1% heat-inactivated Horse Serum) and incubated (forexample for 18 hours at 37° C. in 5% CO₂). Preferably, individualcolonies can be counted up to 80 CFU per aliquot. The following threetest samples can be used as controls: buffer+bacteria+complement;buffer+bacteria+inactivated complement; serum+bacteria+inactivatedcomplement. SBA titers can be straightforwardly calculated using aprogram which processes the data to give a measurement of the dilutionwhich corresponds to 50% of cell killing by a regression calculation.

Animal Protection Assays

Alternatively, the synergistic response may be characterised by theefficacy of the combination of antigens in an animal protection assay.For instance, the assays described in example 12 or 13 may be used.Preferably the number of animals protected by the combination ofantigens is significantly improved compared with using the antigens bythemselves, particularly at suboptimal doses of antigen.

Adhesion Blocking Assay

Alternatively, the immunological response may be characterised by theefficacy of the combination of antigens in an adhesion blocking assay.Preferably the extent of blocking induced by antisera raised against themutant FrpB is significantly improved compared with using antiseraraised against the wild-type FrpB, particularly at suboptimal doses ofantibody.

Polynucleotide

The present invention also provides polynucleotides which code for themutant proteins of the present invention, including variants,derivatives and homologs thereof. Polynucleotides of the invention maycomprise DNA or RNA. They may be single-stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of the invention.

In one embodiment the mutant proteins of the present invention areproduced using any one of the following techniques: site-directedmutagenesis including cassette mutatgenesis, single primer extension, aPCR method of site-directed mutagensis for example the four-primermethod of Higuchi et al (1988) Nucleic Acids Res. 16:7351-67,unidirectional deletion; random mutagenesis; and selection of mutantproteins by phage display.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence codes for a mutant FrpB polypeptide.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology (preferably identity) to the sequences shown in thesequence listing herein. More preferably there is at least 95%, morepreferably at least 98%, homology (preferably identity). Nucleotidehomology comparisons may be conducted as described above. A preferredsequence comparison program is the GCG Wisconsin Bestfit programdescribed above. The default scoring matrix has a match value of 10 foreach identical nucleotide and −9 for each mismatch. The default gapcreation penalty is −50 and the default gap extension penalty is −3 foreach nucleotide.

The present invention also encompasses nucleotide sequences that arecapable of hybridising selectively to the sequences presented herein, orany variant, fragment or derivative thereof, or to the complement of anyof the above. Nucleotide sequences are preferably at least 15nucleotides in length, more preferably at least 20, 30, 40 or 50nucleotides in length.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides of the invention capable of selectively hybridising tothe nucleotide sequences presented herein, or to their complement, willbe generally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides. Preferred polynucleotides of the inventionwill comprise regions homologous to nucleotides which code for conservedregions, preferably at least 80 or 90% and more preferably at least 95%homologous (preferably identical) to these regions.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide of theinvention is found to hybridize to the probe at a level significantlyabove background. The background hybridization may occur because ofother polynucleotides present, for example, in the cDNA or genomic DNAlibrary being screening. In this event, background implies a level ofsignal generated by interaction between the probe and a non-specific DNAmember of the library which is less than 10 fold, preferably less than100 fold as intense as the specific interaction observed with the targetDNA. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm—5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}).

Where the polynucleotide of the invention is double-stranded, bothstrands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other bacterial homologues may be obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Polynucleotides of the invention may be used to produce a primer, e.g. aPCR primer, a primer for an alternative amplification reaction, a probee.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein. Preferred fragments areless than 5000, 2000, 1000, 500 or 200 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes according tothe invention may be produced recombinantly, synthetically, or by anymeans available to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the sequence which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom an animal or human cell, performing a polymerase chain reactionunder conditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector

Refolding Method

The present invention provides a method for promoting the correctfolding/refolding of an FrpB protein or a mutant thereof according tothe present invention, which method involves the use of the detergentSB-12 in an alkaline refolding buffer.

Typically the method of the present invention is used to assist inrefolding recombinantly produced FrpB, which is obtained in an unfoldedor misfolded form. Thus, recombinantly produced proteins may becontacted with the refolding buffer to unfold, refold and/or reactivaterecombinant proteins which are inactive due to misfolding and/or areunfolded as a result of their extraction from the host cells in whichthey were expressed (such as from bacterial inclusion bodies). Such aprocess may also be termed “reconditioning”.

The method of the invention may be employed to maintain the foldedconformation of FrpB, for example during storage, in order to increaseshelf life. Under storage conditions, many proteins lose their activity,as a result of disruption of correct folding. The presence of therefolding buffer of the present invention, reduces or reverses thetendency of proteins to become unfolded and thus greatly increases theshelf life thereof.

The method of the invention may be used to promote the correct foldingof FrpB which, through storage, exposure to denaturing conditions orotherwise, have become misfolded. Thus, the invention may be used torecondition FrpB. For example, FrpB in need of reconditioning may becontacted with the refolding buffer in accordance with the invention.

The present invention also provides a method for altering the structureof an FrpB protein. Structural alterations include folding, unfoldingand refolding. The effect of the alterations is preferably to improvethe yield, specific activity and/or quality of the molecule. This maytypically be achieved by resolubilising, reconditioning and/orreactivating incorrectly folded molecules post-synthesis.

The terms “reconditioning” and “reactivating” thus encompass in vitroprocedures. Particular examples of in vitro procedures may includeprocessing proteins that have been solubilised from cell extracts (suchas inclusion bodies) using strong denaturants such as urea or guanidiumchloride.

The terms “refold”, “reactivate” and “recondition” are not intended asbeing mutually exclusive. For example, an inactive protein, perhapsdenatured using urea, may have an unfolded structure. This inactiveprotein may then be refolded with a refolding buffer of the inventionthereby reactivating it. In some circumstances there may be an increasein the specific activity of the refolded/reactivated protein compared tothe protein prior to inactivation/denaturation: this is termed“reconditioning”.

The molecule is typically an unfolded or misfolded protein which is inneed of folding. Alternatively, however, it may be a folded proteinwhich is to be maintained in a folded state.

The invention envisages at least two situations. A first situation isone in which the protein to be folded is in an unfolded or misfoldedstate, or both. In this case, its correct folding is promoted by themethod of the invention. A second situation is one in which the proteinis substantially already in its correctly folded state, that is all ormost of it is folded correctly or nearly correctly. In this case, themethod of the invention serves to maintain the folded state of theprotein by affecting the folded/unfolded equilibrium so as to favour thefolded state. This prevents loss of activity of an already substantiallycorrectly folded protein. These, and other, eventualities are covered bythe reference to “promoting” the folding of the protein.

As used herein, a protein may be unfolded when at least part of it hasnot yet acquired its correct or desired secondary or tertiary structure.A protein is misfolded when it has acquired at least partially incorrector undesired secondary or tertiary structure. Techniques are known inthe art for assessing protein structure—such as circular dichroism.

The refolding buffer of the present invention comprises3-dimethyldodecylammoniopropanesulfonate (also referred to asN-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and referred toherein as “SB-12”. SB-12 is also referred to as Zwittergent 3-12. SB-12is available from commercial sources, such as Fluka AG. Although SB-12is highly preferred, the use of other salts of SB 12 may also be used,as well as derivatives of SB-12 or molecules related to SB-12. Whilstnot wishing to be bound by any theory, we believe the dilution step inSB-12 (or the presence of SB-12 in general) creates a hydrophobicenvironment for the protein, which is similar to the protein's naturalin vivo environment.

SB-12 is a detergent and the concentration of SB-12 should be at leastabout 0.2% (w/v), since this is the concentration generally required formicelle formation. Thus, the concentration of SB-12 may be about 0.2% toabout 5.0%, about 0.3% to about 4.0%, about 0.4% to about 3.0%, or about0.5% to about 2.0%. Preferably the concentration is about 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%. In an especially preferredembodiment 0.5% SB-12 is used. By “about” it is preferably meant +/−10%of the value of the figure quoted, but is most preferably the exactfigure quoted.

In one embodiment the SB-12 is purified. Conveniently the SB-12 may bepurified before use bypassing a concentrated solution of SB-12 over anA1203 chromatography column, e.g. using in methanol/chloroform (1:1),but any suitable method may be used for purifying the SB-12.

We have found that the dilution of the denatured protein should becarried out in an alkaline environment to maximise the efficiency ofrefolding. Preferably the pH of the refolding buffer is about 11.0.Preferably the alkaline environment is obtained by the use ofethanolamine. In this preferred embodiment the refolding buffercomprises SB-12 and ethanolamine. Conveniently 20 mM ethanolamine isused, but other concentrations, such as 10, 30, 40 or 50 mM, may beuseful.

We have also found that preferably the dilution of the protein may alsobe carried out in the presence of guanidium hydrochloride, NaCl (such as0.4M NaCl) or urea (such as 8M urea), with guanidium hydrochloride beingpreferred. Conveniently 0.4M guanidium hydrochloride is used, but otherconcentrations may be useful. When one of the aforementioned componentsis used during solubilisation, it will be appreciated that there may beno need to add the component again during the refolding stage. Mostpreferably, however, if the protein of the invention is made viainclusion bodies, it is preferably to solubilise the inclusion body withGuanidinium hydrochloride; in such case the refolding buffer need notcontain any denaturant, and the refolded solution may then exchange itsbuffer for 0.3% SB-12 at neutral pH (e.g. Hepes pH 7.5) to stabilise thefolded protein.

We have found that a 1:10 dilution of the FrpB in the refolding bufferis preferred, but other ratios such as 1:20 may be used. Any suitabledilution to effect refolding may be employed readily by a skilledperson.

The FrpB to be processed by the method of the invention is typicallyobtained from cell extracts of host cells expressing recombinant FrpB.Host cells include prokaryotes such as E. coli, yeast and insect cells(the baculovirus system is capable of very high level proteinexpression). Expression of the FrpB in the host cell is preferably athigh levels to maximise yield. Further details on the expression ofrecombinant FrpB are given below.

We have found that the present invention is most efficient when used torefold a mature or mutant mature FrpB protein. By “mature” we include aprotein without a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, and also a protein, but the method can be usedto refold proteins with such sequences. It is common during conventionalpurification techniques to make use of a marker sequence thatfacilitates purification, such as a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilsonet al., Cell 37: 767 (1984). The process of the present invention can beused when such marker sequences are not present.

It is likely that a substantial proportion of the FrpB will be insolubleand consequently techniques to solubilise normally insoluble componentsof the cell extracts (such as inclusion bodies) to maximise extractionof the FrpB will typically be employed. Any conventional technique forpreparation and extraction of the FrpB proteins from inclusion bodiesand their subsequent solubilisation may be employed. Such techniques aredescribed for example in “Current Protocols in Protein Science”published by JA Wiley & Sons. Such techniques generally include:

Cell Lysis—using, for example, a French press or sonication, to releaseinclusion bodies. Typically the cells are placed in a cold buffer suchas a TE buffer prior to lysis. Sonication may be carried out using aBranson sonifier. Sonification may take place in the presence of adetergent, e.g. Brij or Triton. The inclusion bodies in the cell lysatemay then be pelleted using low-speed centrifugation. The cells may bepretreated with lysozyme prior to lysis. The purpose of the pretreatmentis to aid removal of the peptidoglycan and outer membrane proteincontaminants during the washing steps. The lysed cells may be clarifiedby centrifugation and the supernatant discarded.

Inclusion Body Washing—to remove cell wall and other outer membranecomponents contaminants from the inclusion bodies recovered from celllysates. Typically the pellet is resuspended in a wash buffercontaining, e.g. buffer such as TE buffer and/or a detergent such asTriton. The suspension may then be resuspended and the supernatantdiscarded. This process may be repeated until the supernatant is clear.If required, the washed pellets can be frozen for storage.

The amount of recombinant protein in the washed pellet may be estimatedusing the following guidelines: (1) an expression level of 1%corresponds to ˜1 mg recombinant protein per 1 g wet cells. (2) Therecovery of highly aggregated recombinant protein in the washed pelletsis ˜75% that originally present in the cells. (3) About 60% of the totalwashed pellet protein is recombinant-derived. The total amount ofrecombinant protein can be directly determined be measuring the totalprotein concentration or by analysing the washed pellets via SDS-PAGE todetermine the proportions of the protein constituents.

Protein solubilisation—the extracted protein is then extracted from thewashed pellet and defolded using a denaturant which disassociatesprotein-protein interactions and unfolds the protein so that it consistsof unfolded monomers. Denaturants include guanidine:HCl (such as 6Mguanidine:HCl) and/or urea (such as 8M urea). In one embodiment of thepresent invention we conveniently use 8M urea for solubilisation. In apreferred embodiment 6M guanidine:HCl is used. Residual insolublematerial and materials can be typically removed by ultracentrifugation(e.g. 100,000 g× for 1 hr.). The extract may be stored by freezingfollowing pelleting.

Solubilised cell extracts may optionally be partially purified by, forexample, a variety of affinity chromatography techniques prior tocontacting with the refolding buffer according to the method of theinvention.

The solubilised cell extract is then diluted into the refolding bufferin accordance with the present invention. It is typically, but notnecessarily, left stirring at room temperature overnight.

Thus, the starting material for the refolding/reconditioning method ofthe invention is typically denatured proteins in solutions of agentssuch as urea/guanidium chloride. Alternatively, or in addition, solubleprotein samples may be specifically denatured by the addition ofappropriate denaturing agents prior to refolding.

The method of the invention may also employ the use of molecularchaperones.

Chaperones, including chaperoning, are proteins which promote proteinfolding by non-enzymatic means, in that they do not catalyse thechemical modification of any structures in folding proteins, but promotethe correct folding of proteins by facilitating correct structuralalignment thereof. Molecular chaperones are well known in the art,several families thereof being characterised. The invention may employany molecular chaperone molecule, which term includes, for example, themolecular chaperones selected from the following non-exhaustive group:

p90 Calnexin, HSP family, HSP70 family, DNA K, DNAJ, HSP60 family(GroEL), ER-associated chaperones, HSP90, Hsc70, sHsps; SecA; SecB,Trigger factor, zebrafish hsp 47, 70 and 90, HSP 47, GRP 94, Cpn 10,BiP, GRP 78, Clp, FtsH, Ig invariant chain, mitochondrial hsp70, EBP,mitochondrial m-AAA, Yeast Ydj1, Hsp104, ApoE, Syc, Hip, TriC family,CCT, PapD and calmodulin (see WO99/05163 for references).

The method of the present invention may also make use of a foldase. Ingeneral terms, a foldase is an enzyme which participates in thepromotion of protein folding through its enzymatic activity to catalysethe rearrangement or isomerisation of bonds in the folding protein. Theyare thus distinct from a molecular chaperone, which bind to proteins inunstable or non-native structural states and promote correct foldingwithout enzymatic catalysis of bond rearrangement. Many classes offoldase are known, and they are common to animals, plants and bacteria.They include peptidyl prolyl isomerases and thiol/disulphideoxidoreductases. The invention may employ any of the foldases which arecapable of promoting protein folding through covalent bondrearrangement.

At the end of the refolding/reconditioning process, the refolded FrpBmay be desalted by dialysis against a suitable storage buffer and/or theuse of a desalting column into a suitable storage buffer. Suitablebuffers include 25 mM sodium phosphate, 150 mM NaCl and 0.1% PEG 6000(pH 7.4).

In one embodiment of the present invention, following removal of ureaand guanidium hydrochloride, the buffer may be changed from ethanolaminepH11 to Tris-HCl pH7.2 containing the Zwittergent 3-12.

Vectors, Host Cells, Expression Systems

The invention may employ vectors that comprise a polynucleotide whichcodes for at least an FrpB protein and may comprise polynucleotides ofthe present invention which code for a mutant FrpB protein of thepresent invention, host cells that are genetically engineered withvectors of the invention and the production of FrpB proteins byrecombinant techniques. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from DNAconstructs.

Recombinant proteins of the present invention may be prepared byprocesses well known to those skilled in the art from geneticallyengineered host cells comprising expression systems.

For recombinant production of the proteins of the invention, host cellscan be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis,Haemophilus influenzae and Neisseria meningitidis; fungal cells, such ascells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candidaalbicanis and Aspergillus; insect cells such as cells of Drosophila S2and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of agymnosperm or angiosperm.

A great variety of expression systems can be used to produce theproteins of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express aprotein in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed protein. Thesesignals may be endogenous to the protein or they may be heterologoussignals. Proteins of the present invention can be recovered and purifiedfrom recombinant cell cultures by the method of the present invention.

Antibodies

The proteins of the invention can be used as immunogens to produceantibodies immunospecific for such proteins.

In certain preferred embodiments of the invention there are providedantibodies against the FrpB protein of the invention.

Antibodies generated against the proteins of the invention can beobtained by administering the proteins of the invention, orepitope-bearing fragments of either or both, analogues of either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies to proteinsof this invention. Also, transgenic mice, or other organisms or animals,such as other mammals, may be used to express humanized antibodiesimmunospecific to the proteins of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a protein of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-FrpB or from naivelibraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, etal., (1992) Biotechnology 10, 779-783). The affinity of these antibodiescan also be improved by, for example, chain shuffling (Clackson et al.,(1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing FrpB proteins of the invention to purify the proteinsor polynucleotides by, for example, affinity chromatography.

Thus, among others, antibodies against the FrpB protein of the inventionmay be employed to treat infections, particularly bacterial infections.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarily determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

A protein of the present invention can be administered to a recipientwho then acts as a source of immune globulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat Neisserial infection. Hyperimmune globulins of theinvention are particularly useful for treatment or prevention ofNeisserial disease in infants, immune compromised individuals or wheretreatment is required and there is no time for the individual to produceantibodies in response to vaccination.

An additional aspect of the invention is a pharmaceutical compositioncomprising a monoclonal antibody (or fragments thereof; preferably humanor humanised) reactive against the pharmaceutical composition of theinvention, which could be used to treat or prevent infection by Gramnegative bacteria, preferably Neisseria, more preferably Neisseriameningitidis or Neisseria gonorrhoeae and most preferably Neisseriameningitidis serogroup B.

Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE,chimeric antibodies or hybrid antibodies with specificity to two or moreantigens of the invention. They may also be fragments e.g. F(ab′)2,Fab′, Fab, Fv and the like including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein 1975 Nature 256; 495; Antibodies—a laboratory manual Harlow andLane 1988). Alternatively, monoclonal Fv fragments can be obtained byscreening a suitable phage display library (Vaughan T J et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies may be humanised orpart humanised by known methods.

Antagonists and Agonists—Assays and Molecules

The refolded proteins of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

The screening methods may simply measure the binding of a candidatecompound to the protein. Alternatively, the screening method may involvecompetition with a labeled competitor. The screening methods may simplycomprise the steps of mixing a candidate compound with a solutioncontaining a protein of the present invention, to form a mixture,measuring FrpB protein activity in the mixture, and comparing the FrpBprotein activity of the mixture to a standard.

The polynucleotides, proteins and antibodies that bind to and/orinteract with a protein of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or protein in cells. For example, an ELISAassay may be constructed for measuring secreted or cell associatedlevels of protein using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents which mayinhibit or enhance the production of protein (also called antagonist oragonist, respectively) from suitably manipulated cells or tissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of FrpBproteins and polynucleotides, particularly those compounds that arebacteristatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix comprising FrpB protein and alabeled substrate or ligand of such protein is incubated in the absenceor the presence of a candidate molecule that may be a FrpB agonist orantagonist. The ability of the candidate molecule to agonize orantagonize the FrpB protein is reflected in decreased binding of thelabeled ligand or decreased production of product from such substrate.Molecules that bind gratuitously, i.e., without inducing the effects ofFrpB protein are most likely to be good antagonists. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in FrpB protein activity, and bindingassays known in the art.

Another example of an assay for FrpB agonists is a competitive assaythat combines FrpB and a potential agonist with FrpB-binding molecules,recombinant FrpB binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. FrpB can be labeled, such as byradioactivity or a colorimetric compound, such that the number of FrpBmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include, among others, small organic molecules,peptides, proteins and antibodies that bind to a polynucleotide and/orprotein of the invention and thereby inhibit or extinguish its activityor expression. Potential antagonists also may be small organicmolecules, a peptide, a protein such as a closely related protein orantibody that binds the same sites on a binding molecule, such as abinding molecule.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the protein thereby preventing binding tocellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of FrpB.

The invention also provides the use of the protein, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial FrpB proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided FrpB agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual with FrpBprotein of the present invention, or a fragment or variant thereof,adequate to produce antibody and/or T cell immune response to protect(or treat) said individual from infection, particularly bacterialinfection and most particularly Neisseria meningitidis infection. Alsoprovided are methods whereby such immunological response slows bacterialreplication.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a FrpB protein of thepresent invention, wherein the composition comprises a recombinant FrpBof the invention. The immunological response may be used therapeuticallyor prophylactically and may take the form of antibody immunity and/orcellular immunity, such as cellular immunity arising from CTL or CD4+ Tcells.

A FrpB protein, variant or a fragment thereof may be fused withco-protein or chemical moiety which may or may not by itself produceantibodies, but which is capable of producing a fused or modifiedprotein which will have antigenic and/or immunogenic properties, andpreferably protective properties, and optionally may stabilise the FrpB,or render it easier to purify. The co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system ofthe organism receiving the protein.

Also provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the proteins of the invention andimmunostimulatory DNA sequences, such as those described in Sato, Y. etal. Science 273: 352 (1996).

The invention thus also includes a vaccine formulation which comprisesan immunogenic protein of the invention or a fragment or a variantthereof, together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the proteins may be broken down in thestomach, each is preferably administered parenterally, including, forexample, administration that is subcutaneous, intramuscular,intravenous, or intradermal. Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteristaticcompounds and solutes which render the formulation isotonic with thebodily fluid, preferably the blood, of the individual; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The formulation mayalso be administered mucosally, e.g. intranasally.

The vaccine formulation of the invention may also include adjuvantsystems for enhancing the immunogenicity of the formulation. Typicallyaluminium phosphate or aluminium hydroxide may be used. Preferably theadjuvant system raises preferentially a TH1 type of response.

An immune response may be broadly distinguished into two extremecategories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses.

In mice TH1-type responses are often characterised by the generation ofantibodies of the IgG2a subtype, whilst in the human these correspond toIgG1 type antibodies. TH2-type immune responses are characterised by thegeneration of a broad range of immunoglobulin isotypes including in miceIgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p 145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2-type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant,and is preferred. This is known from GB 2220211 (Ribi). Chemically it isa mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains and is manufactured by Ribi Immunochem, Montana. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inEuropean Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).Altenatively, other non-toxic derivatives of LPS may be used.

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (European Patent number 0 689454). 3D-MPL will be present in the range of 10 μg-100 μg preferably25-50 μg per dose wherein the antigen will typically be present in arange 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), or other non-toxic LPS derivative, optionally together with acarrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:13D-MPL: QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In a particularly preferredaspect the antigens in the vaccine composition according to theinvention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating cancers,autoimmune diseases and related conditions. Such a polyvalent vaccinecomposition may include a TH-1 inducing adjuvant as hereinbeforedescribed.

Subunit Composition

The composition of the present invention may be in the form of a subunitcomposition. When the FrpB protein of the invention (whether mutant ornot) is in a folded conformation it is preferably in the form of such asubunit vaccine. Subunit compositions are compositions in which thecomponents have been isolated and purified to at least 50%, preferablyat least 60%, 70%, 80%, 90% pure before mixing the components to formthe antigenic composition.

Subunit compositions may comprise aqueous solutions of water solubleproteins. They may comprise detergent, preferably non-ionic,zwitterionic or ionic detergent in order to solubilise hydrophobicportions of the antigens. They may comprise lipids so that liposomestructures could be formed, allowing presentation of antigens with astructure that spans a lipid membrane. Further details on compositionsis given below.

Outer Membrane Vesicle Preparations

The composition of the present invention may also be in the form of anouter membrane vesicle preparation, particularly when the compositioncomprises the mutant FrpB protein of the present invention.

N. meningitidis serogroup B (menB) excretes outer membrane blebs insufficient quantities to allow their manufacture on an industrial scale.An outer membrane vesicles may also be prepared via the process ofdetergent extraction of the bacterial cells (see for example EP 11243).

The immunogenic composition of the invention may also comprise an outermembrane vesicle preparation having at least two antigens which havebeen recombinantly upregulated. Examples of antigens which would beupregulated in such a outer membrane vesicle preparation in addition tothe FrpB protein of the present invention include; NspA, Hsf, Hap,OMP85, TbpA (high), TbpA (low), LbpA, TbpB, LbpB, PilQ and PldA. Suchpreparations would optionally also comprise either or both of LPSimmunotype L2 and LPS immunotype L3.

The manufacture of bleb preparations from Neisserial strains may beachieved by any of the methods well known to a skilled person.Preferably the methods disclosed in EP 301992, U.S. Pat. No. 5,597,572,EP 11243 or U.S. Pat. No. 4,271,147, Frederikson et al. (NIPH Annals[1991], 14:67-80), Zollinger et al. (J. Clin. Invest. [1979],63:836-848), Saunders et al. (Infect. Immun. [1999], 67:113-119),Drabick et al. (Vaccine [2000], 18:160-172) or WO 01/09350 (Example 8)are used. In general, OMVs are extracted with a detergent, preferablydeoxycholate, and nucleic acids are optionally removed enzymatically.Purification is achieved by ultracentrifugation optionally followed bysize exclusion chromatography. If 2 or more different blebs of theinvention are included, they may be combined in a single container toform a multivalent preparation of the invention (although a preparationis also considered multivalent if the different blebs of the inventionare separate compositions in separate containers which are administeredat the same time [the same visit to a practitioner] to a host). OMVpreparations are usually sterilised by filtration through a 0.2 μmfilter, and are preferably stored in a sucrose solution (e.g. 3%) whichis known to stabilise the bleb preparations.

Upregulation of proteins within outer membrane vesicle preparations maybe achieved by insertion of an extra copy of a gene into the Neisserialstrain from which the OMV preparation is derived. Alternatively, thepromoter of a gene can be exchanged for a stronger promoter in theNeisserial strain from which the OMV preparation is derived. Suchtechniques are described in WO01/09350. If an extra copy of the gene isintroduced, it too can have a non-native strong promoter attached foroverexpression. Upregulation of a protein will lead to a higher level ofprotein being present in OMV compared to the level of protein present inOMV derived from unmodified N. meningitidis (for instance strainH44/76). Preferably the level will be 1.5, 2, 3, 4, 5, 7, 10 or 20 timeshigher.

Where LPS is intended to be an additional antigen in the OMV, a protocolusing a low concentration of extracting detergent (for exampledeoxycholate or DOC) may preferably be used in the OMV preparationmethod so as to preserve high levels of bound LPS whilst removingparticularly toxic, poorly bound LPS. The concentration of DOC used ispreferably 0-0.3% DOC, more preferably 0.05%-0.2% DOC, most preferablyaround 0.1% DOC. “Stronger promoter sequence” refers to a regulatorycontrol element that increases transcription for a gene encoding antigenof interest.

“Upregulating expression” refers to any means to enhance the expressionof an antigen of interest, relative to that of the non-modified (i.e.,naturally occurring) bleb. It is understood that the amount of‘upregulation’ will vary depending on the particular antigen of interestbut will not exceed an amount that will disrupt the membrane integrityof the bleb. Upregulation of an antigen refers to expression that is atleast 10% higher than that of the non-modified bleb. Preferably it is atleast 50% higher. More preferably it is at least 100% (2 fold) higher.Alternatively or additionally, upregulating expression may refer torendering expression non-conditional on metabolic or nutritionalchanges, particularly in the case of FrpB, TbpA, TbpB, LbpA and LbpB. Ingeneral where FrpB is overexpressed in an bleb this may be done byremoving regulatory sequences from the promoter, or by replacement ofthe promoter for a strong, non-regulated promoter such as PorA.

Again for the purpose of clarity, the terms ‘engineering a bacterialstrain to produce less of said antigen’ or down regulation refers to anymeans to reduce the expression of an antigen (or the expression of afunctional gene product) of interest, relative to that of thenon-modified (i.e., naturally occurring bleb), preferably by deletion,such that expression is at least 10% lower than that of the non-modifiedbleb. Preferably it is at least 50% lower and most preferably completelyabsent. If the down regulated protein is an enzyme or a functionalprotein, the downregulation may be achieved by introducing one or moremutations resulting in a 10%, 20%, 50%, 80% or preferably a 100%reduction in enzymatic or functional activity.

The engineering steps required to modulate the expression of Neisserialproteins can be carried out in a variety of ways known to the skilledperson. For instance, sequences (e.g. promoters or open reading frames)can be inserted, and promoters/genes can be disrupted by the techniqueof transposon insertion. For instance, for upregulating a gene'sexpression, a strong promoter could be inserted via a transposon up to 2kb upstream of the gene's initiation codon (more preferably 200-600 bpupstream, most preferably approximately 400 bp upstream). Point mutationor deletion may also be used (particularly for down-regulatingexpression of a gene).

Such methods, however, may be quite unstable or uncertain, and thereforeit is preferred that the engineering step is performed via a homologousrecombination event. Preferably, the event takes place between asequence (a recombinogenic region) of at least 30 nucleotides on thebacterial chromosome, and a sequence (a second recombinogenic region) ofat least 30 nucleotides on a vector transformed within the strain.Preferably the regions are 40-1000 nucleotides, more preferably 100-800nucleotides, most preferably 500 nucleotides). These recombinogenicregions should be sufficiently similar that they are capable ofhybridising to one another under highly stringent conditions.

Methods used to carry out the genetic modification events hereindescribed (such as the upregulation or downregulation of genes byrecombination events and the introduction of further gene sequences intoa Neisserial genome) are described in WO01/09350. Typical strongpromoters that may be integrated in Neisseria are porA, porb, IgtF, Opa,p110, lst, and hpuAB. PorA and PorB are preferred as constitutive,strong promoters. It has been established that the PorB promoteractivity is contained in a fragment corresponding to nucleotides −1 to−250 upstream of the initation codon of porB.

Down Regulation/Removal of Variable and Non-Protective ImmunodominantAntigens

Many surface antigens are variable among bacterial strains and as aconsequence are protective only against a limited set of closely relatedstrains. An aspect of this invention covers outer membrane vesicles ofthe invention in which the expression of other proteins is reduced, or,preferably, gene(s) encoding variable surface protein(s) are deleted.Such deletion results in a bacterial strain producing blebs which, whenadministered in a vaccine, have a stronger potential forcross-reactivity against various strains due to a higher influenceexerted by conserved proteins (retained on the outer membranes) on thevaccinee's immune system. Examples of such variable antigens inNeisseria that may be downregulated in the bleb immunogenic compositionsof the invention include PorA, PorB, Opa.

Other types of gene that could be down-regulated or switched off aregenes which, in vivo, can easily be switched on (expressed) or off bythe bacterium. As outer membrane proteins encoded by such genes are notalways present on the bacteria, the presence of such proteins in thebleb preparations can also be detrimental to the effectiveness of thevaccine for the reasons stated above. A preferred example todown-regulate or delete is Neisseria Opc protein. Anti-Opc immunityinduced by an Opc containing bleb vaccine would only have limitedprotective capacity as the infecting organism could easily become Opc⁻.

For example, these variable or non-protective genes may bedown-regulated in expression, or terminally switched off. This has theadvantage of concentrating the immune system on better antigens that arepresent in low amounts on the outer surface of blebs. By down-regulationit is also meant that surface exposed, variable immunodominant loops ofthe above outer membrane proteins may be altered or deleted in order tomake the resulting outer membrane protein less immunodominant.

Methods for downregulation of expression are disclosed in WO01/09350.Preferred combinations of proteins to be downregulated in the blebimmunogenic compositions of the invention include PorA and OpA; PorA andOpC; OpA and OpC; PorA and OpA and OpC.

Detoxification of LPS

The blebs in the immunogenic compositions of the invention may bedetoxified via methods for detoxification of LPS which are disclosed inWO01/09350. In particular methods for detoxification of LPS of theinvention involve the downregulation of htrB and/or msbB enzymes aredisclosed in WO01/09350. Such methods are preferably combined withmethods of bleb extraction involving low levels of DOC, preferably0-0.3% DOC, more preferably 0.05%-0.2% DOC, most preferably around 0.1%DOC.

Cross-Reactive Polysaccharides

The isolation of bacterial outer-membrane blebs from encapsulatedGram-negative bacteria often results in the co-purification of capsularpolysaccharide. In some cases, this “contaminant” material may proveuseful since polysaccharide may enhance the immune response conferred byother bleb components. In other cases however, the presence ofcontaminating polysaccharide material in bacterial bleb preparations mayprove detrimental to the use of the blebs in a vaccine. For instance, ithas been shown at least in the case of N. meningitidis that theserogroup B capsular polysaccharide does not confer protective immunityand is susceptible to induce an adverse auto-immune response in humans.Consequently, outer membrane vesicles of the invention may be isolatedfrom a bacterial strain for bleb production, which has been engineeredsuch that it is free of capsular polysaccharide. The blebs will then besuitable for use in humans. A particularly preferred example of such ableb preparation is one from N. meningitidis serogroup B devoid ofcapsular polysaccharide.

This may be achieved by using modified bleb production strains in whichthe genes necessary for capsular biosynthesis and/or export have beenimpaired. Inactivation of the gene coding for capsular polysaccharidebiosynthesis or export can be achieved by mutating (point mutation,deletion or insertion) either the control region, the coding region orboth (preferably using the homologous recombination techniques describedabove), or by any other way of decreasing the enzymatic function of suchgenes. Moreover, inactivation of capsular biosynthesis genes may also beachieved by antisense over-expression or transposon mutagenesis. Apreferred method is the deletion of some or all of the Neisseriameningitidis cps genes required for polysaccharide biosynthesis andexport. For this purpose, the replacement plasmid pMF121 (described inFrosh et al. 1990, Mol. Microbiol. 4:1215-1218) can be used to deliver amutation deleting the cps CAD (+galE) gene cluster.

Preferably the siaD gene is deleted, or down-regulated in expression orthe gene product enzymatically inactivated by any other way (themeningococcal siaD gene encodes alpha-2,3-sialyltransferase, an enzymerequired for capsular polysaccharide and LOS synthesis). This mutationis preferred in order to cause minimum disruption to LPS epitopes whichare preferably conserved in the preparations of the invention.

In bleb preparations, particularly in preparations extracted with lowDOC concentrations LPS may be used as an antigen in the immunogeniccomposition of the invention. It is however advantageous todownregulate/delete/inactivate enzymatic function of either the lgtE orpreferably lgtB genes/gene products in order to remove human likelacto-N-neotetraose structures. The Neisserial locus (and sequencethereof) comprising the lgt genes for the biosynthesis of LPSoligosaccharide structure is known in the art (Jennings et alMicorbiology 1999 145; 3013-3021). Downregulation/deletion of lgtB (orfunctional gene product) is preferred since it leaves the LPS protectiveepitope intact. In N. meningitidis serogroup B bleb preparations of theinvention, the downregulation/deletion of both siaD and lgtB ispreferred, leading to a bleb preparation with optimal safety and LPSprotective epitope retention.

Immunogenic composition of the invention may comprise at least, one,two, three, four or five different outer membrane vesicle preparations.Where two or more OMV preparations are included, at least one antigen ofthe invention is upregulated in each OMV. Such OMV preparations may bederived from Neisserial strains of the same species and serogroup orpreferably from Neisserial strains of different class, serogroup,serotype, subserotype or immunotype. For example, an immunogeniccomposition may comprise one or more outer membrane vesiclepreparation(s) which contains LPS of immunotype L2 and one or more outermembrane vesicle preparation which contains LPS of immunotype L3. L2 orL3 OMV preparations are preferably derived from a stable strain whichhas minimal phase variability in the LPS oligosaccharide synthesis genelocus.

Combinations

The immunogenic compositions of the present invention, whether insubunit or outer membrane vesicle form, may also comprise at least oneor more of the following:

-   a. one or more subunit vaccines;-   b. one or more outer membrane vesicles with one or more antigens    upregulated; and-   c. a mixture of a. and b;

Any one of more of a-c may include an FrpB protein of the presentinvention.

The immunogenic compositions of the invention may thus also compriseboth a subunit composition and an outer membrane vesicle.

The outer membrane vesicle preparation may have at least one differentantigen selected from the following list which has been recombinantlyupregulated in an outer membrane vesicle: NspA, Hsf, Hap, OMP85, TbpA(high), TbpA (low), LbpA, TbpB, LbpB, NadA, TspA, TspB, PilQ and PldA;and optionally comprise either or both of LPS immunotype L2 and LPSimmunotype L3.

There are several antigens that are particularly suitable for inclusionin a subunit composition due to their solubility. Examples of suchproteins include; FhaB, NspA, passenger domain of Hsf, passenger domainof Hap, OMP85, FrpA, FrpC, TbpB, LbpB, PilQ.

Neisserial infections progress through several different stages. Forexample, the meningococcal life cycle involve nasopharyngealcolonisation, mucosal attachment, crossing into the bloodstream,multiplication in the blood, induction of toxic shock, crossing theblood/brain barrier and multiplication in the cerebrospinal fluid and/orthe meninges. Different molecules on the surface of the bacterium willbe involved in different steps of the infection cycle. By targeting theimmune response against an effective amount of a combination ofparticular antigens, involved in different processes of Neisserialinfection, a Neisserial vaccine with surprisingly high efficacy can beachieved.

In particular, combinations of certain Neisserial antigens fromdifferent classes with the FrpB protein of the invention can elicit animmune response which protects against multiple stages of infection.Such combinations of antigens can surprisingly lead to synergisticallyimproved vaccine efficacy against Neisserial infection where more thatone function of the bacterium is targeted by the immune response in anoptimal fashion. Some of the further antigens which can be included areinvolved in adhesion to host cells, some are involved in ironacquisition, some are autotransporters and some are toxins.

The efficacy of vaccines can be assessed through a variety of assays.Protection assays in animal models are well known in the art.Furthermore, serum bactericidal assay (SBA) is the most commonly agreedimmunological marker to estimate the efficacy of a meningococcal vaccine(Perkins et al. J Infect Dis. 1998, 177:683-691).

Some combinations of antigens can lead to improved protection in animalmodel assays and/or synergistically higher SBA titres. Without wishingto be bound by theory, such synergistic combinations of antigens areenabled by a number of characteristics of the immune response to theantigen combination. The antigens themselves are usually surface exposedon the Neisserial cells and tend to be conserved but also tend not to bepresent in sufficient quantity on the surface cell for an optimalbactericidal response to take place using antibodies elicited againstthe antigen alone. Combining the antigens of the invention can result ina formulation eliciting an advantageous combination of bactericidalantibodies which interact with the Neisserial cell beyond a criticalthreshold. At this critical level, sufficient antibodies of sufficientquality bind to the surface of the bacterium to allow efficient killingby complement and much higher bactericidal effects are seen as aconsequence. As serum bactericidal assays (SBA) closely reflect theefficacy of vaccine candidates, the attainment of good SBA titres by acombination of antigens is a good indication of the protective efficacyof a vaccine containing that combination of antigens.

An additional advantage of the invention is that the combination of theantigens of the invention from different families of proteins in animmunogenic composition will enable protection against a wider range ofstrains.

The invention thus also relates to immunogenic compositions comprising aplurality of proteins selected from at least two different categories ofprotein, having different functions within Neisseria. Examples of suchcategories of proteins are adhesins, autotransporter proteins, toxinsand Fe acquisition proteins. The vaccine combinations of the inventionshow surprising improvement in vaccine efficacy against homologousNeisserial strains (strains from which the antigens are derived) andpreferably also against heterologous Neisserial strains.

In particular, the invention provides immunogenic compositions thatcomprise at least one, two, three, four five, six, seven, eight, nine orten different additional Neisseria antigens (to FrpB) selected from atleast one, two, three, four or five groups of proteins selected from thefollowing:

-   at least one Neisserial adhesin selected from the group consisting    of FhaB, Hsf, NspA, NadA, PilC, Hap, MafA, MafB, Omp26, NMB0315,    NMB0995 and NMB1119;-   at least one Neisserial autotransporter selected from the group    consisting of Hsf, Hap, IgA protease, AspA and NadA;-   at least one Neisserial toxin selected from the group consisting of    FrpA, FrpC, FrpA/C, VapD, NM-ADPRT, and either or both of LPS    immunotype L2 and LPS immunotype L3;-   at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA high, TbpA low, TbpB high, TbpB low, LbpA,    LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp,    NMB0964 and NMB0293; and-   at least one Neisserial membrane associated protein, preferably    outer membrane protein, selected from the group consisting of PldA,    TspA, FhaC, NspA, TbpA(high), TbpA(low), LbpA, HpuB, TdfH, PorB,    HimD, HisD, GNA1870, OstA, HlpA, MltA, NMB 1124, NMB 1162, NMB 1220,    NMB 1313, NMB 1953, HtrA, TspB, PilQ and OMP85.    and preferably:-   a. at least one Neisserial adhesin selected from the group    consisting of FhaB, Hsf and NadA;-   b. at least one Neisserial autotransporter selected from the group    consisting of Hsf, Hap and NadA;-   c. at least one Neisserial toxin selected from the group consisting    of FrpA, FrpC, and either or both of LPS immunotype L2 and LPS    immunotype L3;-   d. at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA, TbpB, LbpA and LbpB; and-   e. at least one Neisserial outer membrane protein selected from the    group consisting of TspA, TspB, NspA, PilQ, OMP85, and PldA.

Preferably the first four (and most preferably all five) groups ofantigen are represented in the immunogenic composition of the invention.

As previously mentioned where a protein is specifically mentionedherein, it is preferably a reference to a native, full-length proteinbut it may also encompass antigenic fragments thereof (particularly inthe context of subunit vaccines). These are fragments containing orcomprising at least 10 amino acids, preferably 20 amino acids, morepreferably 30 amino acids, more preferably 40 amino acids or mostpreferably 50 amino acids, taken contiguously from the amino acidsequence of the protein. In addition, antigenic fragments denotesfragments that are immunologically reactive with antibodies generatedagainst the Neisserial proteins or with antibodies generated byinfection of a mammalian host with Neisseria. Antigenic fragments alsoincludes fragments that when administered at an effective dose, elicit aprotective immune response against Neisserial infection, more preferablyit is protective against N. meningitidis and/or N. gonorrhoeaeinfection, most preferably it is protective against N. meningitidisserogroup B infection.

Also included in the invention are recombinant fusion proteins ofNeisserial proteins of the invention, or fragments thereof. These maycombine different Neisserial proteins or fragments thereof in the sameprotein. Alternatively, the invention also includes individual fusionproteins of Neisserial proteins or fragments thereof, as a fusionprotein with heterologous sequences such as a provider of T-cellepitopes, or viral surface proteins such as influenza virushaemagglutinin, tetanus toxoid, diphtheria toxoid, CRM197.

Addition Antigens of the Invention

NMB references refer to reference numbers to sequences which can beaccessed from www.neisseria.org.

1. Adhesins

Adhesins include FhaB (WO98/02547), NadA (J. Exp. Med (2002) 195:1445;NMB 1994), Hsf also known as NhhA (NMB 0992) (WO99/31132), Hap (NMB1985)(WO99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB0643) (Annu Rev Cell Dev Biol. 16; 423-457 (2000); Nature Biotech 20;914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC(Mol.

Microbiol. 1997, 23; 879-892). These are proteins that are involved inthe binding of Neisseria to the surface of host cells. Hsf is an exampleof an adhesin, as well as being an autotranporter protein. Immunogeniccompositions of the invention may therefore include combinations of Hsfand other autotransporter proteins where Hsf contributes in its capacityas an adhesin. These adhesins may be derived from Neisseria meningitidisor Neisseria gonorrhoeae or other Neisserial strains. The invention alsoincludes other adhesins from Neisseria.

FhaB

This antigen has been described in WO98/02547 SEQ ID NO 38 (nucleotides3083-9025)—see also NMB0497. The present inventors have found FhaB to beparticularly effectively at inducing anti-adhesive antibodies alone andin particular with other antigens of the invention. Although full lengthFhaB could be used, the inventors have found that particular C-terminaltruncates are surprisingly at least as effective and preferably evenmore effective in terms of cross-strain effect. Such truncates have alsobeen advantageously shown to be far easier to clone. FhaB truncates ofthe invention typically correspond to the N-terminal two-thirds of theFhaB molecule, preferably the new C-terminus being situated at position1200-1600, more preferably at position 1300-1500, and most preferably atposition 1430-1440. Specific embodiments have the C-terminus at 1433 or1436. Accordingly such FhaB truncates of the invention and vaccinescomprising such truncates are preferred components of the combinationimmunogenic compositions of the invention. The N-terminus may also betruncated by up to 10, 20, 30, 40 or 50 amino acids.

2. Autotransporter Proteins

Autotransporter proteins typically are made up of a signal sequence, apassenger domain and an anchoring domain for attachment to the outermembrane. Examples of autotransporter proteins include Hsf (WO99/31132)(NMB 0992), HMW, Hia (van Ulsen et al Immunol. Med. Microbiol. 2001 32;53-64), Hap (NMB 1985) (WO99/55873; van Ulsen et al Immunol. Med.Microbiol. 2001 32; 53-64), UspA, UspA2, NadA (NMB 1994) (Comanducci etal J. Exp. Med. 2002 195; 1445-1454), AspA (Infection and Immunity 2002,70(8); 4447-4461; NMB 1029), Aida-1 like protein, SSh-2 and Tsh. NadA(J. Exp. Med (2002) 195:1445) is another example of an autotransporterproteins, as well as being an adhesin. Immunogenic compositions of theinvention may therefore include combinations of NadA and adhesins whereNadA contributes in its capacity as an autotransporter protein. Theseproteins may be derived from Neisseria meningitidis or Neisseriagonorrhoeae or other Neiserial strians. The invention also includesother autotransporter proteins from Neisseria.

Hsf

Hsf has a structure that is common to autotransporter proteins. Forexample, Hsf from N. meningitidis strain H44/76 consists of a signalsequence made up of amino acids 1-51, a head region at the aminoterminus of the mature protein (amino acids 52-479) that is surfaceexposed and contains variable regions (amino acids 52-106, 121-124,191-210 and 230-234), a neck region (amino acids 480-509), a hydrophobicalpha-helix region (amino acids 518-529) and an anchoring domain inwhich four transmembrane strands span the outer membrane (amino acids539-591).

Although full length Hsf may be used in immunogenic compositions of theinvention, various Hsf truncates and deletions may also beadvantageously used depending on the type of vaccine.

Where Hsf is used in a subunit vaccine, it is preferred that a portionof the soluble passenger domain is used; for instance the completedomain of amino acids 52 to 479, most preferably a conserved portionthereof, for instance the particularly advantageous sequence of aminoacids 134 to 479. Preferred forms of Hsf may be truncated so as todelete variable regions of the protein disclosed in WO01/55182.Preferred variants would include the deletion of one, two, three, four,or five variable regions as defined in WO01/55182. The above sequencesand those described below, can be extended or truncated by up to 1, 3,5, 7, 10 or 15 amino acids at either or both N or C termini.

Preferred fragments of Hsf therefore include the entire head region ofHsf, preferably containing amino acids 52-473. Additional preferredfragments of Hsf include surface exposed regions of the head includingone or more of the following amino acid sequences; 52-62, 76-93,116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306, 328-338,362-391, 408-418, 430-440 and 469-479.

Where Hsf is present in an outer membrane vesicle preparation, it may beexpressed as the full-length protein or preferably as an advantageousvariant made up of a fusion of amino acids 1-51 and 134-591 (yielding amature outer membrane protein of amino acid sequence 134 to theC-terminus). Preferred forms of Hsf may be truncated so as to deletevariable regions of the protein disclosed in WO01/55182. Preferredvariants would include the deletion of one, two, three, four, or fivevariable regions as defined in WO01/55182. Preferably the first andsecond variable regions are deleted. Preferred variants would deleteresidues from between amino acid sequence 52 through to 237 or 54through to 237, more preferably deleting residues between amino acid 52through to 133 or 55 through to 133. The mature protein would lack thesignal peptide.

Hap

Computer analysis of the Hap-like protein from Neisseria meningitidisreveals at least three structural domains. Considering the Hap-likesequence from strain H44/76 as a reference, Domain 1, comprisingamino-acid 1 to 42, encodes a sec-dependant signal peptidecharacteristic of the auto-transporter family, Domain 2, comprisingamino-acids 43 to 950, encode the passenger domain likely to be surfaceexposed and accessible to the immune system, Domain 3, comprisingresidues 951 to the C-terminus (1457), is predicted to encode abeta-strands likely to assemble into a barrel-like structure and to beanchored into the outer-membrane. Since domains 2 is likely to besurface-exposed, well conserved (more than 80% in all strain tested) andcould be produced as subunit antigens in E. coli, it represents aninteresting vaccine candidates. Since domains 2 and 3 are likely to besurface-exposed, are well conserved (Pizza et al. (2000), Science 287:1816-1820), they represent interesting vaccine candidates. Domain 2 isknown as the passenger domain.

Immunogenic compositions of the invention may comprise the full-lengthHap protein, preferably incorporated into an OMV preparation.Immunogenic compositions of the invention may also comprise thepassenger domain of Hap which in strain H44/76 is composed of amino acidresidues 43-950. This fragment of Hap would be particularlyadvantageously used in a subunit composition of the invention. The abovesequence for the passenger domain of Hap can be extended or truncated byup to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both Nor C termini.

3. Iron Acquisition Proteins

Iron aquisition proteins include TbpA (NMB 0461) (WO92/03467, U.S. Pat.No. 5,912,336, WO93/06861 and EP586266), TbpB (NMB 0460) (WO93/06861 andEP586266), LbpA (NMB 1540) (Med Microbiol (1999) 32:1117), LbpB (NMB1541)(WO/99/09176), HpuA (U73112.2) (Mol Microbiol. 1997, 23; 737-749),HpuB (NC_(—)003116.1) (Mol Microbiol. 1997, 23; 737-749), P2086 alsoknown as XthA (NMB 0399) (13^(th) International Pathogenic NeisseriaConference 2002), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB1206), Lipo28 also known as GNA2132 (NMB 2132), Sibp (NMB 1882), HmbR,HemH, Bcp (NMB 0750), Iron (III) ABC transporter-permease protein(Tettelin et al Science 287; 1809-1815 2000), Iron (III) ABCtransporter—periplasmic (Tettelin et al Science 287; 1809-1815 2000),TonB-dependent receptor (NMB 0964 and NMB 0293) (Tettelin et al Science287; 1809-1815 2000) and transferrin binding protein related protein(Tettelin et al Science 287; 1809-1815 2000). These proteins may bederived from Neisseria meningitidis, Neisseria gonorrhoeae or otherNeisserial strains. The invention also includes other iron aquisitionproteins from Neisseria.

TbpA

TbpA interacts with TbpB to form a protein complex on the outer membraneof Neisseria, which binds transferrin. Structurally, TbpA contains anintracellular N-terminal domain with a TonB box and plug domain,multiple transmembrane beta strands linked by short intracellular andlonger extracellular loops.

Two families of TbpB have been distinguished, having a high molecularweight and a low molecular weight respectively. High and low molecularweight forms of TbpB associate with different families of TbpA which aredistinguishable on the basis of homology. Despite being of similarmolecular weight, they are known as the high molecular weight and lowmolecular weight families because of their association with the high orlow molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.100; 51, 1993). The terms TbpA(high) and TbpA(low) are used to refer tothese two forms of TbpA, and similarly for TbpB. Immunogeniccompositions of the invention may comprise TbpA and TbpB from serogroupsA, B, C, Y and W-135 of N. meningitidis as well as iron acquisitionproteins from other bacteria including N. gonorrhoeae. Transferrinbinding proteins TbpA and TbpB have also been referred to as Tbp1 andTbp2 respectively (Cornelissen et al Infection and Immunity 65; 822,1997).

TbpA contains several distinct regions. For example, in the case of TbpAfrom N. meningitidis strain H44/76, the amino terminal 186 amino acidsform an internal globular domain, 22 beta strands span the membrane,forming a beta barrel structure. These are linked by short intracellularloops and larger extracellular loops.

Extracellular loops 2, 3 and 5 have the highest degree of sequencevariability and loop 5 is surface exposed. Loops 5 and 4 are involved inligand binding.

Preferred fragments of TbpA include the extracellular loops of TbpA.Using the sequence of TbpA from N. meningitidis strain H44/76, theseloops correspond to amino acids 200-202 for loop 1, amino acids 226-303for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop4, amino acids 512-576 for loop 5, amino acids 609-625 for loop 6, aminoacids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids872-903 for loop 11. The corresponding sequences, after sequencealignment, in other Tbp proteins would also constitute preferredfragments. Most preferred fragments would include amino acid sequencesconstituting loop 2, loop 3, loop 4 or loop 5 of Tbp.

Where the immunogenic compositions of the invention comprise TbpA, it ispreferable to include both TbpA(high) and TbpA (low).

Although TbpA is preferably presented in an OMV vaccine, it may also bepart of a subunit vaccine. For instance, isolated iron acquisitionproteins which could be introduced into an immmunogenic composition ofthe invention are well known in the art (WO00/25811). They may beexpressed in a bacterial host, extracted using detergent (for instance2% Elugent) and purified by affinity chromatography or using standardcolumn chromatography techniques well known to the art (Oakhill et alBiochem J. 2002 364; 613-6).

Where TbpA is presented in an OMV vaccine, its expression can beupregulated by genetic techiques discussed herein or in WO 01/09350, ormay preferably be upregulated by growth of the parent strain under ironlimitation conditions. This process will also result in the upregulationof variable iron-regulated proteins, particularly wild-type FrpB whichmay become immunodominant and it is therefore advantageous todownregulate the expression of (and preferably delete the genesencoding) such proteins (particularly wild-type FrpB) as described in WO01/09350, or remove its immunodominant loops as described above, toensure that the immunogenic composition of the invention elicits animmune response against antigens present in a wide range of Neisserialstrains. If wild-type FrpB is deleted, an additional copy of a nonimmunodominant mutant FrpB gene may be introduced into the cell. It ispreferred to have both TbpA(high) and TbpA(low) present in theimmunogenic composition and this is preferably achieved by combiningOMVs derived from two strains, expressing the alternative forms of TbpA.

4. Toxins

Toxins include FrpA (NMB 0585; NMB 1405), FrpA/C (see below fordefinition), FrpC (NMB 1415; NMB 1405) (WO92/01460), NM-ADPRT (NMB 1343)(13^(th) International Pathogenic Neisseria Conference 2002 Masignani etal p135), VapD (NMB 1753), lipopolysaccharide (LPS; also calledlipooligosaccharide or LOS) immunotype L2 and LPS immunotype L3. FrpAand FrpC contain a region which is conserved between these two proteinsand a preferred fragment of the proteins would be a polypeptidecontaining this conserved fragment, preferably comprising amino acids227-1004 of the sequence of FrpA/C. These antigens may be derived fromNeisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The invention also includes other toxins from Neisseria.

In an alternative embodiment, toxins may include antigens involved inthe regulation of toxicity, for example OstA which functions in thesynthesis of lipopolysaccharides.

FrpA and FrpC

Neisseria meningitidis encodes two RTX proteins, referred to as FrpA &FrpC secreted upon iron limitation (Thompson et al., (1993) J.Bacteriol. 175:811-818; Thompson et al., (1993) Infect. Immun.61:2906-2911). The RTX (Repeat ToXin) protein family have in common aseries of 9 amino acid repeat near their C-termini with the consensus:Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa. (LXGGXGN_(/D)DX). The repeatsin E. coli HlyA are thought to be the site of Ca2+ binding. Asrepresented in FIG. 4, meningococcal FrpA and FrpC proteins, ascharacterized in strain FAM20, share extensive amino-acid similarity intheir central and C-terminal regions but very limited similarity (ifany) at the N-terminus. Moreover, the region conserved between FrpA andFrpC exhibit some polymorphism due to repetition (13 times in FrpA and43 times in FrpC) of a 9 amino acid motif.

Immunogenic compositions of the invention may comprise the full lengthFrpA and/or FrpC or preferably, a fragment comprising the sequenceconserved between FrpA and FrpC. The conserved sequence is made up ofrepeat units of 9 amino acids. Immunogenic compositions of the inventionwould preferably comprise more that three repeats, more than 10 repeats,more than 13 repeats, more than 20 repeats or more than 23 repeats.

Such truncates have advantageous properties over the full lengthmolecules, and vaccines comprising such antigens are preferred for beingincorporated in the immunogenic compositions of the invention.

Sequences conserved between FrpA and FrpC are designated FrpA/C andwhereever FrpA or FrpC forms a constituent of immunogenic compositionsof the invention, FrpA/C could be advantageously used. Amino acids277-1004 of the FrpA sequence is the preferred conserved region. Theabove sequence can be extended or truncated by up to 1, 3, 5, 7, 10, 15,20, 25, or 30 amino acids at either or both N or C termini.

LPS

LPS (lipopolysaccharide, also known as LOS—lipooligosaccharide) is theendotoxin on the outer membrane of Neisseria. The polysaccharide moietyof the LPS is known to induce bactericidal antibodies.

Heterogeneity within the oligosaccharide moiety of the LPS generatesstructural and antigenic diversity among different neisserial strains(Griffiss et al. Inf. Immun. 1987; 55: 1792-1800). This has been used tosubdivide meningococcal strains into 12 immunotypes (Scholtan et al. JMed Microbiol 1994, 41:236-243). Immunotypes L3, L7, & L9 areimmunologically identical and are structurally similar (or even thesame) and have therefore been designated L3,7,9 (or, for the purposes ofthis specification, generically as “L3”). Meningococcal LPS L3,7,9 (L3),L2 and L5 can be modified by sialylation, or by the addition of cytidine5′-monophosphate-N-acetylneuraminic acid. Although L2, L4 and L6 LPS aredistinguishable immunologically, they are structurally similar and whereL2 is mentioned herein, either L4 or L6 may be optionally substitutedwithin the scope of the invention. See M. P. Jennings et al,Microbiology 1999, 145, 3013-3021 and Mol Microbiol 2002, 43:931-43 forfurther illustration of LPS structure and heterogeneity.

Where LPS, preferably meningococcal LPS, is included in a vaccine of theinvention, preferably and advantageously either or both of immunotypesL2 and L3 are present. LPS is preferably presented in an outer membranevesicle (preferably where the vesicle is extracted with a low percentagedetergent, more preferably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%,0.08-0.15% or 0.1%, most preferably deoxycholate [DOC]) but may also bepart of a subunit vaccine. LPS may be isolated using well knownprecedure including the hot water-phenol procedure (Wesphal and JannMeth. Carbo. Chem. 5; 83-91 1965). See also Galanos et al. 1969, Eur JBiochem 9:245-249, and Wu et al. 1987, Anal Bio Chem 160:281-289. LPSmay be used plain or conjugated to a source of T-cell epitopes such astetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membraneproteins. Techniques for conjugating isolated LOS are also known (seefor instance EP 941738 incorporated by reference herein).

Where LOS (in particular the LOS of the invention) is present in a blebformulation the LOS is preferably conjugated in situ by methods allowingthe conjugation of LOS to one or more outer membrane proteins alsopresent on the bleb preparation (e.g. PorA or PorB in meningococcus).

This process can advantageously enhance the stability and/orimmunogenicity (providing T-cell help) and/or antigenicity of the LOSantigen within the bleb formulation—thus giving T-cell help for theT-independent oligosaccharide immunogen in its most protectiveconformation—as LOS in its natural environment on the surface ofmeningococcal outer membrane. In addition, conjugation of the LOS withinthe bleb can result in a detoxification of the LOS (the Lipid A portionbeing stably buried in the outer membrane thus being less available tocause toxcity). Thus the detoxification methods mentioned herein ofisolating blebs from htrB⁻ or msbB⁻ mutants, or by adding non toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] to the composition (see WO 93/14115, WO 95/03327,Velucchi et al (1997) J Endotoxin Res 4: 1-12, and EP 976402 for furtherdetails of non-toxic peptide functional equivalents of polymyxinB—particularly the use of the peptide SAEP 2 (of sequence KTKCKFLKKCwhere the 2 cysteines form a disulphide bridge)) may not be required(but which may be added in combination for additional security). Thusthe inventors have found that a composition comprising blebs wherein LOSpresent in the blebs has been conjugated in an intra-bleb fashion toouter membrane proteins also present in the bleb can form the basis of avaccine for the treatment or prevention of diseases caused by theorganism from which the blebs have been derived, wherein such vaccine issubstantially non-toxic and is capable of inducing a T-dependentbactericidal response against LOS in its native environment.

Such bleb preparations may be isolated from the bacterial in question(see WO 01/09350), and then subjected to known conjugation chemistriesto link groups (e.g. NH₂ or COOH) on the oligosaccharide portion of LOSto groups (e.g. NH₂ or COOH) on bleb outer membrane proteins.Cross-linking techniques using glutaraldehyde, formaldehyde, orglutaraldehyde/formaldehyde mixes may be used, but it is preferred thatmore selective chemistries are used such as EDAC or EDAC/NHS (J. V.Staros, R. W. Wright and D. M. Swingle. Enhancement byN-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176). Other conjugationchemistries or treatments capable of creating covalent links between LOSand protein molecules that could be used are described in EP 941738.

Preferably the bleb preparations are conjugated in the absence ofcapsular polysaccharide. The blebs may be isolated from a strain whichdoes not produce capsular polysaccharide (naturally or via mutation asdescribed below), or may be purified from most and preferably allcontaminating capsular polysaccharide. In this way, the intra-bleb LOSconjugation reaction is much more efficient.

Preferably more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of theLOS present in the blebs is cross-linked/conjugated.

Intrableb conjugation should preferably incorporate 1, 2 or all 3 of thefollowing process steps: conjugation pH should be greater than pH 7.0,preferably greater than or equal to pH 7.5 (most preferably under pH 9);conditions of 1-5% preferably 2-4% most preferably around 3% sucroseshould be maintained during the reaction; NaCl should be minimised inthe conjugation reaction, preferably under 0.1M, 0.05M, 0.01M, 0.005M,0.001M, and most preferably not present at all. All these processfeatures make sure that the blebs remain stable and in solutionthroughout the conjugation process.

The EDAC/NHS conjugation process is a preferred process for intra-blebconjugation. EDAC/NHS is preferred to formalydehyde which can cross-linkto too high an extent thus adversely affecting filterability. EDACreacts with carboxylic acids (such as KDO in LOS) to create anactive-ester intermediate. In the presence of an amine nucleophile (suchas lysines in outer membrane proteins such as PorB), an amide bond isformed with release of an isourea by-product. However, the efficiency ofan EDAC-mediated reaction may be increased through the formation of aSulfo-NHS ester intermediate. The Sulfo-NHS ester survives in aqueoussolution longer than the active ester formed from the reaction of EDACalone with a carboxylate. Thus, higher yields of amide bond formationmay be realized using this two-stage process. EDAC/NHS conjugation isdiscussed in J. V. Staros, R. W. Wright and D. M. Swingle. Enhancementby N-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176. Preferably 0.01-5 mgEDAC/mg bleb is used in the reaction, more preferably 0.05-1 mg EDAC/mgbleb. The amount of EDAC used depends on the amont of LOS present in thesample which in turn depends on the deoxycholate (DOC) % used to extractthe blebs. At low % DOC (e.g. 0.1%), high amounts of EDAC are used (1mg/mg and beyond), however at higher % DOC (e.g. 0.5%), lower amounts ofEDAC are used (0.025-0.1 mg/mg) to avoid too much inter-blebcrosslinking.

A preferred process of the invention is therefore a process forproducing intra-bleb conjugated LOS (preferably meningococcal)comprising the steps of conjugating blebs in the presence of EDAC/NHS ata pH between pH 7.0 and pH 9.0 (preferably around pH 7.5), in 1-5%(preferably around 3%) sucrose, and optionally in conditionssubstantially devoid of NaCl (as described above), and isolating theconjugated blebs from the reaction mix.

The reaction may be followed on Western separation gels of the reactionmixture using anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show theincrease of LOS molecular weight for a greater proportion of the LOS inthe blebs as reaction time goes on.

Yields of 99% blebs can be recovered using such techniques.

EDAC was found to be an excellent intra-bleb cross-linking agent in thatit cross-linked LOS to OMP sufficiently for improved LOS T-dependentimmunogenicity, but did not cross link it to such a high degree thatproblems such as poor filterability, aggregation and inter-blebcross-linking occurred. The morphology of the blebs generated is similarto that of unconjugated blebs (by electron microscope). In addition, theabove protocol avoided an overly high cross-linking to take place (whichcan decrease the immunogenicity of protective OMPs naturally present onthe surface of the bleb e.g. TbpA or Hsf).

It is preferred that the meningococcal strain from which the blebs arederived is a mutant strain that cannot produce capsular polysaccharide(in particular siaD⁻). It is also preferred that immunogeniccompositions effective against meningococcal disease comprise both an L2and and L3 bleb, wherein the L2 and L3 LOS are both conjugated to blebouter membrane proteins. Furthermore, it is preferred that the LOSstructure within the intra-bleb conjugated bleb is consistent with ithaving been derived from an lgtE⁻ or, preferably, lgtB⁻ meningococcalstrain. Most preferably immunogenic compositions compriseintrableb-conjugated blebs: derived from a mutant meningococcal strainthat cannot produce capsular polysaccharide and is lgtB⁻; comprising L2and L3 blebs derived from mutant meningococcal strains that cannotproduce capsular polysaccharide; comprising L2 and L3 blebs derived frommutant meningococcal strains that are lgtB⁻; or most preferablycomprising L2 and L3 blebs derived from mutant meningococcal strainsthat cannot produce capsular polysaccharide and are lgtB⁻.

Typical L3 meningococcal strain that can be used for the presentinvention is H44/76 menB strain. A typical L2 strain is the B16B6 menBstrain or the 39E meningococcus type C strain.

As stated above, the blebs of the invention have been detoxified to adegree by the act of conjugation, and need not be detoxified anyfurther, however further detoxification methods may be used foradditional security, for instance using blebs derived from ameningococcal strain that is htrB⁻ or msbB⁻ or adding a non-toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] (preferably SEAP 2) to the bleb composition (asdescribed above).

In the above way meningococcal blebs and immunogenic compositionscomprising blebs are provided which have as an important antigen LOSwhich is substantially non-toxic, devoid of autoimmunity problems, has aT-dependent character, is present in its natural environment, and iscapable of inducing a bactericidal antibody response against more than90% of meningococcal strains (in the case of L2+L3 compositions).

5. Integral Outer Membrane Proteins

Other categories of Neisserial proteins may also be candidates forinclusion in the Neisserial vaccines of the invention and may be able tocombine with other antigens in a surprisingly effective manner. Membraneassociated proteins, particularly integral membrane proteins and mostadvantageously outer membrane proteins, especially integral outermembrane proteins may be used in the compositions of the presentinvention. An example of such a protein is PldA also known as Omp1A (NMB0464) (WO00/15801) which is a Neisserial phospholipase outer membraneprotein. Further examples are TspA (NMB 0341) (Infect. Immun. 1999, 67;3533-3541) and TspB (T-cell stimulating protein) (WO 00/03003; NMB 1548,NMB 1628 or NMB 1747). Further examples include PilQ (NMB 1812)(WO99/61620), OMP85—also known as D15—(NMB 0182) (WO00/23593), NspA(U52066) (WO96/29412), FhaC (NMB 0496 or NMB 1780), PorB (NMB 2039)(Mol. Biol. Evol. 12; 363-370, 1995), HpuB (NC_(—)003116.1), TdfH (NMB1497) (Microbiology 2001, 147; 1277-1290), OstA (NMB 0280), MltA alsoknown as GNA33 and Lipo3O (NMB0033), HtrA (NMB 0532; WO 99/55872), HimD(NMB 1302), HisD (NMB 1581), GNA 1870 (NMB 1870), HlpA (NMB 1946), NMB1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TbpA (NMB 0461)(WO92/03467) (see also above under iron acquisition proteins) and LbpA(NMB 1541).

OMP85

OMP85/D15 is an outer membrane protein having a signal sequence, aN-terminal surface-exposed domain and an integral membrane domain forattachment to the outer membrane. Immunogenic compositions of theinvention may also comprise the full length OMP85, preferably as part ofan OMV preparation. Fragments of OMP85 may also be used in immunogeniccompositions of the invention, in particularly, the N terminalsurface-exposed domain of OMP85 made up of amino acid residues 1-475 or50-475 is preferably incorporated into a subunit component of theimmunogenic compositions of the invention. The above sequence for the Nterminal surface-exposed domain of OMP85 can be extended or truncated byup to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both Nor C termini. It is preferred that the signal sequence is omitted fromthe OMP85 fragment.

OstA

OstA functions in the synthesis of lipopolysaccharides and may beconsidered to be a regulator of toxicity. OstA may alternatively beincluded in the toxin category where the toxin category is broadened tocontain regulators of toxicity as well as toxins.

Preferably the subunit composition comprises FrpB of the presentinvention together with:

-   i) at least one further antigen selected from the following list:    FhaB, passenger domain of Hsf, passenger domain of Hap, NadA,    N-terminal surface exposed domain of OMP85, FrpA, FrpC, FrpA/C,    TpbA, TbpB, LpbA, LbpB, PldA, PilQ, NspA and either or both of LPS    immunotype L2 and LPS immunotype L3; and/or-   ii) at least a Neisserial (preferably meningococcal) outer membrane    vesicle (OMV) preparation. Preferably the OMV preparation has at    least one antigen (more preferably 2, 3, 4 or 5) selected from the    following list which has been recombinantly upregulated in the outer    membrane vesicle: NspA, Hsf, Hap, OMP85, TbpA (high), TbpA (low),    LbpA, TbpB, LbpB, PilQ and PldA; and optionally comprising either or    both of LPS immunotype L2 and LPS immunotype L3.

When i) is present the additional antigen is preferably selected fromone or more of the groups of proteins given above.

In another embodiment the outer membrane vesicle of the presentinvention has at least one further antigen (more preferably 2, 3, 4 or5) is recombinantly upregulated in the outer membrane vesicle andselected from the following list: NspA, Hsf, Hap, OMP85, TbpA (high),TbpA (low), LbpA, TbpB, LbpB, PilQ and PldA; and optionally comprisingeither or both of LPS immunotype L2 and LPS immunotype L3. This outermembrane vesicle may be used with one or more further outer membranevesicles in which has at least one further antigen (more preferably 2,3, 4 or 5) is recombinantly upregulated in the outer membrane vesicleand selected from the following list: FrpB, NspA, Hsf, Hap, OMP85, TbpA(high), TbpA (low), LbpA, TbpB, LbpB, PilQ and PldA; and optionallycomprising either or both of LPS immunotype L2 and LPS immunotype L3.

The immunogenic compositions of the invention may comprise antigens(proteins, LPS and polysaccharides) derived from Neisseria meningitidisserogroups A, B, C, Y, W-135 or Neisseria gonorrhoeae.

Further Combinations

The immunogenic composition of the invention may further comprisebacterial capsular polysaccharides or oligosaccharides. The capsularpolysaccharides or oligosaccharides may be derived from one or more of:Neisseria meningitidis serogroup A, C, Y, and/or W-135, Haemophilusinfluenzae b, Streptococcus pneumoniae, Group A Streptococci, Group BStreptococci, Staphylococcus aureus and Staphylococcus epidermidis.

A further aspect of the invention are vaccine combinations comprisingthe antigenic composition of the invention with other antigens which areadvantageously used against certain disease states including thoseassociated with viral or Gram positive bacteria.

In one preferred combination, the antigenic compositions of theinvention are formulated with 1, 2, 3 or preferably all 4 of thefollowing meningococcal capsular polysaccharides or oligosaccharideswhich may be plain or conjugated to a protein carrier: A, C, Y or W-135.Preferably the immunogenic compositions of the invention are formulatedwith A and C; or C; or C and Y. Such a vaccine containing proteins fromN. meningitidis serogroup B may be advantageously used as a globalmeningococcus vaccine.

In a further preferred embodiment, the antigenic compositions of theinvention, preferably formulated with 1, 2, 3 or all 4 of the plain orconjugated meningococcal capsular polysaccharides or oligosaccharides A,C, Y or W-135 (as described above), are formulated with a conjugated H.influenzae b capsular polysaccharide (or oligosaccharides), and/or oneor more plain or conjugated pneumococcal capsular polysaccharides (oroligosaccharides) (for instance those described below). Optionally, thevaccine may also comprise one or more protein antigens that can protecta host against Streptococcus pneumoniae infection. Such a vaccine may beadvantageously used as a global meningitis vaccine.

In a still further preferred embodiment, the immunogenic composition ofthe invention is formulated with capsular polysaccharides oroligosaccharides derived from one or more of Neisseria meningitidis,Haemophilus influenzae b, Streptococcus pneumoniae, Group AStreptococci, Group B Streptococci, Staphylococcus aureus orStaphylococcus epidermidis. The pneumococcal capsular polysaccharide oroligosaccharide antigens are preferably selected from serotypes 1, 2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V,14, 18C, 19F and 23F). A further preferred embodiment would contain thePRP capsular polysaccharides or oligosaccharides of Haemophilusinfluenzae. A further preferred embodiment would contain the Type 5,Type 8 or 336 capsular polysaccharides of Staphylococcus aureus. Afurther preferred embodiment would contain the Type I, Type II or TypeIII capsular polysaccharides of Staphylococcus epidermidis. A furtherpreferred embodiment would contain the Type Ia, Type Ic, Type II or TypeIII capsular polysaccharides of Group B streptococcus. A furtherpreferred embodiment would contain the capsular polysaccharides of GroupA streptococcus, preferably further comprising at least one M proteinand more preferably multiple types of M protein.

Such capsular polysaccharides or oligosaccharides of the invention maybe unconjugated or conjugated to a carrier protein such as tetatustoxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197,pneumolysin, Protein D (U.S. Pat. No. 6,342,224). The polysaccharide oroligosaccharide conjugate may be prepared by any known couplingtechnique. For example the polysaccharide can be coupled via a thioetherlinkage. This conjugation method relies on activation of thepolysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated polysaccharide may thus becoupled directly or via a spacer group to an amino group on the carrierprotein. Preferably, the cyanate ester is coupled with hexane diamineand the amino-derivatised polysaccharide is conjugated to the carrierprotein using heteroligation chemistry involving the formation of thethioether linkage. Such conjugates are described in PCT publishedapplication WO93/15760 Uniformed Services University.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508. A further method involves the coupling of acyanogen bromide activated polysaccharide derivatised with adipic acidhydrazide (ADH) to the protein carrier by Carbodiimide condensation (ChuC. et al Infect. Immunity, 1983 245 256).

Preferred pneumococcal proteins antigens are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus. Most preferably, the protein is a toxin,adhesin, 2-component signal tranducer, or lipoprotein of Streptococcuspneumoniae, or fragments thereof. Particularly preferred proteinsinclude, but are not limited to: pneumolysin (preferably detoxified bychemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990Jul. 11; 18(13): 4010 “Comparison of pneumolysin genes and proteins fromStreptococcus pneumoniae types 1 and 2.”, Mitchell et al. BiochimBiophys Acta 1989 Jan. 23; 1007(1): 67-72 “Expression of the pneumolysingene in Escherichia coli: rapid purification and biologicalproperties.”, WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO99/03884 (NAVA)]; PspA and transmembrane deletion variants thereof (U.S.Pat. No. 5,804,193—Briles et al.); PspC and transmembrane deletionvariants thereof (WO 97/09994—Briles et al); PsaA and transmembranedeletion variants thereof (Berry & Paton, Infect Immun 1996 December;64(12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumoniae”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate -dehydrogenase(Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, (EP0837130) and adhesin 18627, (EP 0834568). Further preferred pneumococcalprotein antigens are those disclosed in WO 98/18931, particularly thoseselected in WO 98/18930 and PCT/US99/30390.

The immunogenic composition/vaccine of the invention may also optionallycomprise outer membrane vesicle preparations made from other Gramnegative bacteria, for example Moraxella catarrhalis or Haemophilusinfluenzae.

Compositions, Kits and Administration

As previously mentioned the invention provides compositions comprising aFrpB protein for administration to a cell or to a multicellularorganism.

An immunogenic composition is a composition comprising at least oneantigen which is capable of generating an immune response whenadministered to a host. Preferably, such immunogenic preparations arecapable of generating a protective immune response against Neisserial,preferably Neisseria meningitidis and/or Neisseria gonorrhoeaeinfection.

The invention also relates to compositions comprising a proteindiscussed herein or their agonists or antagonists. The proteins of theinvention may be employed in combination with a non-sterile or sterilecarrier or carriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to an individual.Such compositions comprise, for instance, a media additive or atherapeutically effective amount of a protein of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Proteins and other compounds of the invention may be employed alone orin conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a protein or other compounds of the present invention canbe formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgement of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Preferred features and embodiment of the present invention will now bedescribed further with reference to the following Examples:

EXAMPLES Example 1

Refolding of FrpB

FrpB is the most abundant iron-limitation-inducible outer-membraneprotein of Neisseria meningitidis. The high expression levels togetherwith considerable sequence conservation between strains and its abilityto induce bactericidal antibodies make it an interesting vaccinecandidate for the prevention of meningococcal disease. However, theprotein does contain some variability, which is mostly confined to twolarge extracellular loops, L5 and L7, respectively (van der Ley et al.1996).

In an attempt to direct the immune response to the more conserved partsof the protein, a deletion of 28 amino acid residues was created in loopL7. A second mutant was created which besides the 28 amino acidsdeletion in loop L7, contains an additional deletion of 24 amino acidsin loop L5. By removing L5 in addition to L7, we aim to construct anFrpB protein that consists of only very-well conserved domains of FrpB,which could potentially induce broad-spectrum bactericidal antibodies.FrpB and FrpB loop-deletion mutants were produced in inclusion bodies inEscherichia coli and subsequently refolded in vitro. Refolding wassuccessful and immune responses against these proteins may be analysed.

I: Construction of Genes Encoding FrpB and Genes Encoding FrpB LoopDeletion Mutants

The gene encoding Neisseria meningitidis H44/76 FrpB was cloned withoutsignal sequence in pET11a resulting in plasmid pET11a-FrpB. An FrpB loopdeletion mutant was prepared by using the four-primer PCR method tocreate a deletion of 28 amino acids in the extracellular loop L7. Bythis way, the encoded protein will lack amino acidstteekngqkvdkpmeqqmkdradedtvh from loop L7. This gene was transferred topET11a without signal sequence, resulting in plasmid pET11a-FrpBΔ28.This construct was used to create plasmid pET11a-FrpBΔ7Δ5. This plasmidencodes a protein, which besides the 28 amino acids deletion in loop L7,contains an additional deletion of amino acids qhrgirtvreeftvgdkssrinidfrom loop L5. The above uses the old model of loop nomenclature of FIG.6.

II: Overexpression and In vitro Refolding of FrpB, FrpBΔ28 and FrpBΔ7Δ5

FrpB, FrpBΔ28 and FrpBΔ7Δ5 were produced in Escherichia coli BL21, afterwhich inclusion bodies were isolated. The inclusion bodies weresubsequently solubilized in 8 M urea. Refolding was performed with 1:10dilution of the solubilized material in 20 mM ethanolamine pH 11, 0.5%Zwittergent 3-12 (SB-12), 0.4 M guanidium hydrochloride. Refolding wasperformed at room temperature for 16 hrs. Urea and guanidiumhydrochloride were removed and buffer was changed from ethanolamine pH11 to Tris-HCl pH 7.2. Refolded forms were detected by semi-native polyacrylamide gel electrophoresis. Electrophoresis was performed with gelslacking SDS.

The running buffer contained 0.02% SDS, and 0.075% (cold samples) or 2%SDS (boiled samples) was used in the sample buffer. Samples were keptcold or boiled for 5 minutes, after which electrophoresis was performedat constant current of 10 mA on ice.

III: Results

The urea-denatured proteins were diluted 1:10 to ethanolamine pH 11,0.5% Zwittergent 12, 0.4 M guanidium hydrochloride. After 16 hrs.samples were desalted and buffer was exchanged for Tris-HCl pH 7.2. Therunning buffer contained 0.02% SDS, and 0.075% (cold samples) or 2% SDS(boiled samples) was used in the sample buffer. Samples were kept coldor boiled for 5 minutes, after which electrophoresis was performed atconstant current of 10 mA on ice.

The results obtained from semi-native gel electrophoresis show foldedmonomers of FrpB, FrpBΔ28 and FrpBΔ7Δ5. These folded forms can beobserved as proteins with higher electrophoretic mobility as compared tothe denatured protein. It is also clear that in vitro refolded FrpB aswell as FrpBΔ28 can assemble into oligomeric complexes, which is a keycharacteristic of native FrpB. The ability of FrpBΔ7Δ5 to formoligomeric complexes is clearly impaired, however the presence of foldedmonomers indicates correct folding.

These correctly folded proteins may be be used to study the ability ofthese proteins to induce synthesis of protective antibodies againstNeisseria meningitidis. Correct folding of these proteins is consideredcrucial for obtaining a protective immune response, as nativeconformational epitopes are more likely to be present.

Example 2

Improved Protocol for Refolding FrpB, and Mutants Thereof

Inclusion bodies were solubilized in 6 M Guanidium hydrochloride andsubsequently quickly (vortex) diluted 1:20 in 20-27 mM Ethanolamine (noHCl or NaOH added)+0.5% SB-12 (Zwittergent 3-12). The next day, thebuffer was exchanged for Hepes pH 7.5+0.3% SB-12 to stabilize theproteins.

This improved protocol prevents the occurrence of aggregates duringrefolding. In FIG. 10A a picture of in vitro folded FrpB according tothe new protocol is shown, together with its corresponding CD spectrumrecorded on a Jasco-810 spectropolarimeter [average of 30 scans] (FIG.10B).

Example 3

Evaluation of the Immune Response Against In vitro Folded FrpB (Mutants)

Immunization experiments were performed with in vitro folded FrpB,FrpBΔ28 and FrpBΔ5/Δ3 (new loop nomenclature—see FIG. 9). Sera wereanalysed by ELISA with in vitro folded FrpB as antigen.

These sera were analysed with a Western-blot on the parent strain and 16heterologous stains (FIG. 10A). FrpB was detected in all strains,indicating recognition of conserved epitopes. Improved detection ofstrains BNCV and 2996 (*) was observed with the antiserum raised againstFrpBΔ5/Δ3.

Conclusion: Good responses were observed from all three immunizations,resulting in serum that was cross-reactive between stains. Furthermore,the enhanced recognition of strains 2996 and BNCV, suggests that theimmune response can be re-directed towards different domains of theprotein by immunization with a deletion mutant.

Example 4

Immunization With OMVs Carrying Overexpressed FrpB (Mutants) or WithRefolded Wild-Type FrpB

To analyse the immune response against OMVs carrying the FrpB (mutant)proteins, the proteins of interest (FrpB, FrpBΔ28, and FrpBΔ5/Δ3 [usingthe new FIG. 9 loop nomenclature]) were overproduced in meningococci(pFP10 derived expression in CE2001 (H44/76 PorA(−)). A vaccine wasproduced according to Fredriksen et al. (NIPH Ann. 1991 Dec.;14(2):67-79; discussion 79-80). Immunization experiments weresubsequently performed with the bleb vaccines as well as with refoldedwild-type FrpB of the invention to obtain sera for SBA experimentsagainst Nmen H44/76.

The preparations were tested in the OF1 mouse model. Animals, 10 pergroup, received three injections by the IM route on Day 0, 21 and 35.Per injection, mice received 5 μg of antigen formulated in adjuvant(Al³⁺ salts+3DMPL). Blood samples were taken 7 days after the second andthe third injection.

Pooled Post II and Post III sera as well as individual post III serawere tested in SBA on homologous (H44/76) and heterologous MenB strainsas well as on a H44/76 FrpB KO strain. Specific anti FrpB antibodies byELISA on pooled mice-sera 7PII and 7PIII Mid-point Titers Group Antigen7PII 7PIII 1 Frpb WT DOC extracts 373 542 2 FrpBD28 DOC extracts 304 5323 FrpBD5/D3 DOC bleb 212 423 4 Frpb WT refolded 3406 4837 5 (−) 25 25

The WT FrpB refolded and in blebs as well as mutants induced theproduction of anti-FrpB Abs (see ELISA table above).

Wild-type FrpB in a bleb, and wild-type refolded FrpB produced highbactericidal antibody titres against H44/76. The use of the H44/76 FrpBKO strain demonstrated that these bactericidal Abs are directed againstFrpB. Blebs carrying FrpBΔ28 and FrpBΔ5/Δ3 produced low bactericidalantibody titres. Although blebs carrying FrpB, FrpBΔ28 and FrpBΔ5/Δ3induced low bactericidal titres against a heterologous MenB strainM97250687, FrpBΔ28 blebs induced a higher SBA than the other two (with atitre over 1/100 dilution).

Example 5

Epitope Display in FrpB Loop Positions

As stated previously, sequence variation in FrpB is mostly confined totwo domains of the protein, corresponding to the proposed loop L3 andloop L5 of the new model (FIG. 9). Of these two loops, loop L5 waspreviously shown to be highly immunogenic and bactericidal antibodiesare directed against this part of the protein. To make use of thetolerance of variation in this loop and its ability to inducebactericidal antibodies in the wild-type configuration, this part of theprotein was chosen to serve as an insertion site of foreign epitopes.

As proof of principle, a very well studied epitope of PorA from H44/76was selected to function as the foreign epitope. The high variableregion corresponding to loop L5 of FrpB (new model)(TTEEKNGQKVDKPMEQQMKDRADEDTVH) was removed and replaced by a shortsequence consisting of two restriction sites (MunI and NheI) with ashort sequence encoding a PorA 1.7,16 fragment from loop 4 [FrpB-C1containing QLKDTNNNAS, sequence corresponding to loop 4 or PorA areunderlined, QL and AS flanking the PorA sequence are encoded by therestriction sites incorporated in the carrier protein].

The flanking restriction sites were subsequently used to insert anoligonucleotide encoding a larger part of loop 4[FrpB-C2-QLKSAYTPAYYTKDTNNNLTLVPAVVAS]. Both genes were subsequentlytransferred without signal sequence to pET11a (Novagen) plasmids.Protein expression was performed in Escherichia coli BL21. Expression ofthese constructs resulted in inclusion bodies, which were isolated. Theinclusion bodies were subsequently solubilized and in vitro foldedaccording to the protocol described earlier. A Western blot as well as adot-blot assay (FIG. 12) was performed with the monoclonal antibodyMN5C11G (1:100,000) (Toropainen et al 2001 Microb Pathog 30:139), whichrecognizes the minimal epitope KDTNNN of P1.7,16.

Both chimeric proteins were successfully refolded in vitro and detectionof the proteins with the PorA antibody indicated correct insertion ofthe foreign fragments. Strikingly, folded forms of the chimeric proteinsreacted more efficiently as compared with denatured protein, suggestingcorrect folding of the foreign epitopes in FrpB.

Example 6

Mimotope Display in FrpB Loop Positions

FrpB loops are also ideal for displaying peptide mimotopes (for instanceof meningococcal LOS). Displaying mimotopes in a correctly foldedcontext allows an immune response against LOS, for example, to begenerated without having the toxic effects of LOS present in thevaccine.

Mab216 is a monoclonal antibody that recognizes an epitope of the innercore LOS. By biopanning on a library of cyclized heptamer peptides withthis Mab, a mimetic peptide was identified in the art (Ian Feavers).

Six amino acids of the peptide were inserted into FrpB loop 5 (new loopnomenclature of FIG. 9) and the chimera was over expressed (pFP10plasmid) in H44/76 Neisseria in an LOS negative context (lpxA Knock Outmutant). See sequence of resulting protein below. OMVs were purified andinjected into mice (OF1 female 6-8 weeks old, 3 injections DO-21-28 by1M route; 5 μg of blebs in ALOH/3DMPL were administered and seracollected on day 41). By Western Blot, Mab 216 was found to recognizethe FrpB-mimotope.

Sequences:

The following pages show the sequence of FrpBΔ28 and FrpBΔ5/Δ3 [new loopnomenclature] (which contains a point mutation resulting in oneamino-acid change). The sequences depicted here correspond to the geneswhich are present in the pFP10 plasmid. Therefore, the signal sequenceis still present. For expression of the proteins in inclusion bodies inE. coli, the corresponding genes without their signal sequences wereused. For the constructs without signal sequence (pET11a), the “G” atposition 110 corresponds to the final nucleotide of the newly createdATG.

FrpBΔ28: Amino acids which are deleted in double mutant are shown inBOLD-UNDERLINED.MNTPLFRLSLLSLTLAAGFAHAAENNAKVVLDTVTVKGDRQGSKIRTNIVTLQQKDESTATDMRELLKEEPSIDFGGGNGTSQFLTLRGMGQNSVDIKVDNAYSDSQILYHQGRFIVDPALVKVVSVQKGAGSASAGIGATNGAIIAKTVDAQDLLKGLDKNWGVRLNSGFASNEGVSYGASVFGKEGNFDGLFSYNRNDEKDYEAGKGFRNVNGGKTVPYSALDKRSYLAKIGTTFGDDDHRIVLSHMKD QHRGIRTVREEFTVGDKSSRINID RQAPAYRETTQSNTNLAYTGKNLGFVEKLDANAYVLEKERYSADDSGTGYAGNVKGPNHTRITTRGANFNFDSRLAEQTLLKYGINYRHQEIKPQAFLNSKFSIPAYKLSNPTKTDTGVYVEAIHDIGDFTLTGGLRYDRFKVKTHDGKTVSSSNLNPSFGVIWQPHEHWSFSASHNYASRSPRLYDALQTHGKRGIISIADGTKAERARNTEIGFNYNDGTFAANGSYFWQTIKDALANPQNRHDSVAVREAVNAGYIKNHGYELGASYRTGGLTAKVGVSHSKPRFYDTHKDKLLSANPEFGAQVGRTWTASLAYRFQNPNLEIGWRGRYVQKATGSILAAGQKDRKGNLENVVRKGFGVNDVFANWKPLGKDTLNVNLSVNNVFNKFYYPHSQRWTNTLPGVGRDV RLGVNYKF

FrpBΔ5/Δ3: Point mutation results in amino-acid change K to R in loop 6(BOLD-UNDERLINED)MNTPLFRLSLLSLTLAAGFAHAAENNAKVVLDTVTVKGDRQGSKIRTNIVTLQQKDESTATDMRELLKEEPSIDFGGGNGTSQFLTLRGMGQNSVDIKVDNAYSDSQILYHQGRFIVDPALVKVVSVQKGAGSASAGIGATNGAIIAKTVDAQDLLKGLDKNWGVRLNSGFASNEGVSYGASVFGKEGNFDGLFSYNRNDEKDYEAGKGFRNVNGGKTVPYSALDKRSYLAKIGTTFGDDDHRIVLSHMKDRQAPAYRETTQSNTNLAYTGKNLGFVEKLDANAYVLEKERYSADDSGTGYAGNVKGPNHTRITTRGANFNFDSRLAEQTLLKYGINYRHQEIKPQAFLNSKFSIPAYKLSNPTKTDTGVYVEAIHDIGDFTLTGGLRYDRFKVKTHDG RTVSSSNLNPSFGVIWQPHEHWSFSASHNYASRSPRLYDALQTHGKRGIISIADGTKAERARNTEIGFNYNDGTFAANGSYFWQTIKDALANPQNRHDSVAVREAVNAGYIKNHGYELGASYRTGGLTAKVGVSHSKPRFYDTHKDKLLSANPEFGAQVGRTWTASLAYRFQNPNLEIGWRGRYVQKATGSILAAGQKDRKGNLENVVRKGFGVNDVFANWKPLGKDTLNVNLSVNNVFNKFYYPHSQRWTNTLPGVGRDVRLGVNYKF

FrpBΔ28: START IS INDICATED IN BOLD AND UNDERLINEDGGCCGCTTGGATTTACTTCCCTTCATACTCAAGAGGACGATTGA ATG AATACCCCATTGTTCCGTCTCAGCCTGCTCTCGCTCACACTTGCGGCAGGTTTTGCCCACGCGGCAGAAAATAATGCCAAGGTCGTACTGGATACCGTTACTGTAAAAGGCGACCGCCAAGGCAGCAAAATCCGTACCAACATCGTTACGCTGCAACAAAAAGACGAAAGCACCGCAACCGATATGCGCGAACTCTTAAAAGAAGAGCCGTCCATCGATTTCGGCGGCGGCAACGGCACGTCCCAATTCCTGACGCTGCGCGGCATGGGTCAGAACTCTGTCGACATCAAGGTGGACAACGCCTATTCCGACAGCCAAATCCTTTACCACCAAGGCAGATTTATTGTCGATCCCGCTTTGGTTAAAGTCGTTTCCGTACAAAAAGGCGCGGGTTCCGCCTCTGCCGGTATCGGCGCGACCAACGGCGCGATCATCGCCAAAACCGTCGATGCCCAAGACCTGCTCAAAGGCTTGGATAAAAACTGGGGCGTGCGCCTCAACAGCGGCTTTGCCAGCAACGAAGGCGTAAGCTACGGCGCAAGCGTATTCGGAAAAGAGGGCAACTTCGACGGCTTGTTCTCTTACAACCGCAACGATGAAAAAGATTACGAAGCCGGCAAAGGTTTCCGCAATGTCAACGGCGGCAAAACCGTACCGTACAGCGCGCTGGACAAACGCAGCTACCTCGCCAAAATCGGAACAACCTTCGGCGACGACGACCACCGCATCGTGTTGAGCCACATGAAAGACCAACACCGGGGCATCCGCACTGTGCGTGAAGAATTTACCGTCGGCGACAAAAGTTCACGGATAAATATTGACCGCCAAGCCCCTGCTTACCGCGAAACTACCCAATCCAACACCAACTTGGCGTACACGGGTAAAAACCTGGGCTTTGTCGAAAAACTGGATGCCAACGCCTATGTGTTGGAAAAAGAACGCTATTCCGCCGATGACAGCGGCACCGGCTACGCAGGCAATGTAAAAGGCCCCAACCATACCCGAATCACCACTCGTGGTGCGAACTTCAACTTCGACAGCCGCCTTGCCGAACAAACCCTGTTGAAATACGGTATCAACTACCGCCATCAGGAAATCAAACCGCAAGCATTTTTGAACTCGAAATTCTCCATCCCCGCCTACAAACTTTCCAACCCGACCAAAACCGATACCGGCGTATATGTTGAAGCCATTCACGACATCGGCGATTTCACGCTGACCGGCGGGCTGCGTTACGACCGCTTCAAGGTGAAAACCCATGACGGCAAAACCGTTTCAAGCAGCAACCTTAACCCGAGTTTCGGTGTGATTTGGCAGCCGCACGAACACTGGAGCTTCAGCGCGAGCCACAACTACGCCAGCCGCAGCCCGCGCCTGTATGACGCGCTGCAAACCCACGGTAAACGCGGCATCATCTCGATTGCCGACGGCACAAAAGCCGAACGCGCGCGCAATACCGAAATCGGCTTCAACTACAACGACGGCACGTTTGCCGCAAACGGCAGCTACTTCTGGCAGACCATCAAAGACGCGCTTGCCAATCCGCAAAACCGCCACGACTCTGTCGCCGTCCGTGAAGCCGTCAATGCCGGTTACATCAAAAACCACGGTTACGAATTGGGCGCGTCCTACCGCACCGGCGGCCTGACTGCCAAAGTCGGCGTCAGCCACAGCAAACCGCGCTTTTACGATACGCACAAAGACAAGCTGTTGAGCGCGAATCCTGAATTTGGCGCACAAGTCGGCCGCACTTGGACGGCCTCCCTTGCCTACCGCTTCCAAAATCCGAATCTGGAAATCGGCTGGCGCGGCCGTTATGTTCAAAAAGCTACGGGTTCGATATTGGCGGCAGGTCAAAAAGACCGCAAAGGCAACTTGGAAAACGTTGTACGCAAAGGTTTCGGTGTGAACGATGTCTTCGCCAACTGGAAACCGCTGGGCAAAGACACGCTCAATGTCAATCTTTCGGTTAACAACGTGTTCAACAAGTTCTACTATCCGCACAGCCAACGCTGGACCAATACCCTGCCGGGCGTGGGACGTGATGTACGCT TGGGCGTGAACTACAAGTTCTAAAACGCACGACGT

FrpBΔ5/Δ3: START AND POINT MUTATION ARE INDICATED IN BOLD + UNDERLINEDGGCCGCTTGGATTTACTTCCCTTCATACTCAAGAGGACGATTGA ATG AATACCCCATTGTTCCGTCTCAGCCTGCTCTCGCTCACACTTGCGGCAGGTTTTGCCCACGCGGCAGAAAATAATGCCAAGGTCGTACTGGATACCGTTACTGTAAAAGGCGACCGCCAAGGCAGCAAAATCCGTACCAACATCGTTACGCTGCAACAAAAAGACGAAAGCACCGCAACCGATATGCGCGAACTCTTAAAAGAAGAGCCGTCCATCGATTTCGGCGGCGGCAACGGCACGTCCCAATTCCTGACGCTGCGCGGCATGGGTCAGAACTCTGTCGACATCAAGGTGGACAACGCCTATTCCGACAGCCAAATCCTTTACCACCAAGGCAGATTTATTGTCGATCCCGCTTTGGTTAAAGTCGTTTCCGTACAAAAAGGCGCGGGTTCCGCCTCTGCCGGTATCGGCGCGACCAACGGCGCGATCATCGCCAAAACCGTCGATGCCCAAGACCTGCTCAAAGGCTTGGATAAAAACTGGGGCGTGCGCCTCAACAGCGGCTTTGCCAGCAACGAAGGCGTAAGCTACGGCGCAAGCGTATTCGGAAAAGAGGGCAACTTCGACGGCTTGTTCTCTTACAACCGCAACGATGAAAAAGATTACGAAGCCGGCAAAGGTTTCCGCAATGTCAACGGCGGCAAAACCGTACCGTACAGCGCGCTGGACAAACGCAGCTACCTCGCCAAAATCGGAACAACCTTCGGCGACGACGACCACCGCATCGTGTTGAGCCACATGAAAGACCGCCAAGCCCCTGCTTACCGCGAAACTACCCAATCCAACACCAACTTGGCGTACACGGGTAAAAACCTGGGCTTTGTCGAAAAACTGGATGCCAACGCCTATGTGTTGGAAAAAGAACGCTATTCCGCCGATGACAGCGGCACCGGCTACGCAGGCAATGTAAAAGGCCCCAACCATACCCGAATCACCACTCGTGGTGCGAACTTCAACTTCGACAGCCGCCTTGCCGAACAAACCCTGTTGAAATACGGTATCAACTACCGCCATCAGGAAATCAAACCGCAAGCATTTTTGAACTCGAAATTCTCCATCCCCGCCTACAAACTTTCCAACCCGACCAAAACCGATACCGGCGTATATGTTGAAGCCATTCACGACATCGGCGATTTCACGCTGACCGGCGGGCTGCGTTACGACCGCTTCAAGGTGAAAACCCATGACGGCAGAACCGTTTCAAGCAGCAACCTTAACCCGAGTTTCGGTGTGATTTGGCAGCCGCACGAACACTGGAGCTTCAGCGCGAGCCACAACTACGCCAGCCGCAGCCCGCGCCTGTATGACGCGCTGCAAACCCACGGTAAACGCGGCATCATCTCGATTGCCGACGGCACAAAAGCCGAACGCGCGCGCAATACCGAAATCGGCTTCAACTACAACGACGGCACGTTTGCCGCAAACGGCAGCTACTTCTGGCAGACCATCAAAGACGCGCTTGCCAATCCGCAAAACCGCCACGACTCTGTCGCCGTCCGTGAAGCCGTCAATGCCGGTTACATCAAAAACCACGGTTACGAATTGGGCGCGTCCTACCGCACCGGCGGCCTGACTGCCAAAGTCGGCGTCAGCCACAGCAAACCGCGCTTTTACGATACGCACAAAGACAAGCTGTTGAGCGCGAATCCTGAATTTGGCGCACAAGTCGGCCGCACTTGGACGGCCTCCCTTGCCTACCGCTTCCAAAATCCGAATCTGGAAATCGGCTGGCGCGGCCGTTATGTTCAAAAAGCTACGGGTTCGATATTGGCGGCAGGTCAAAAAGACCGCAAAGGCAACTTGGAAAACGTTGTACGCAAAGGTTTCGGTGTGAACGATGTCTTCGCCAACTGGAAACCGCTGGGCAAAGACACGCTCAATGTCAATCTTTCGGTTAACAACGTGTTCAACAAGTTCTACTATCCGCACAGCCAACGCTGGACCAATACCCTGCCGGGCGTGGGACGTGATGTACGCTTGGGCGTGAACTACAAGTTCTAAAACGCACGACGT

FrpB-mimo (start and mimotope insert are in bold and underlined)TGTTAATATAAATAAAAATAATTATTAATTATTTTTCTTATCCTGCCAAATCTTAACGGTTTGGATTTACTTCCCTTCATACTCAAGAGGACGATTGA ATG AATACCCCATTGTTCCGTCTCAGCCTGCTCTCGCTCACACTTGCGGCAGGTTTTGCCCACGCGGCAGAAAATAATGCCAAGGTCGTACTGGATACCGTTACTGTAAAAGGCGACCGCCAAGGCAGCAAAATCCGTACCAACATCGTTACGCTGCAACAAAAAGACGAAAGCACCGCAACCGATATGCGCGAACTCTTAAAAGAAGAGCCGTCCATCGATTTCGGCGGCGGCAACGGCACGTCCCAATTCCTGACGCTGCGCGGCATGGGTCAGAACTCTGTCGACATCAAGGTGGACAACGCCTATTCCGACAGCCAAATCCTTTACCACCAAGGCAGATTTATTGTCGATCCCGCTTTGGTTAAAGTCGTTTCCGTACAAAAAGGCGCGGGTTCCGCCTCTGCCGGTATCGGCGCGACCAACGGCGCGATCATCGCCAAAACCGTCGATGCCCAAGACCTGCTCAAAGGCTTGGATAAAAACTGGGGCGTGCGCCTCAACAGCGGCTTTGCCAGCAACGAAGGCGTAAGCTACGGCGCAAGCGTATTCGGAAAAGAGGGCAACTTCGACGGCTTGTTCTCTTACAACCGCAACGATGAAAAAGATTACGAAGCCGGCAAAGGTTTCCGCAATGTCAACGGCGGCAAAACCGTACCGTACAGCGCGCTGGACAAACGCAGCTACCTCGCCAAAATCGGAACAACCTTCGGCGACGACGACCACCGCATCGTGTTGAGCCACATGAAAGACCAACACCGGGGCATCCGCACTGTGCGTGAAGAATTTACCGTCGGCGACAAAAGTTCACGGATAAATATTGACCGCCAAGCCCCTGCTTACCGCGAAACTACCCAATCCAACACCAACTTGGCGTACACGGGTAAAAACCTGGGCTTTGTCGAAAAACTGGATGCCAACGCCTATGTGTTGGAAAAAGAACGCTATTCCGCCGATGACAGCGGCACCGGCTACGCAGGCAATGTAAAAGGCCCCAACCATACCCGAATCACCACTCGTGGTGCGAACTTCAACTTCGACAGCCGCCTTGCCGAACAAACCCTGTTGAAATACGGTATCAACTACCGCCATCAGGAAATCAAACCGCAAGCATTTTTGAACTCGAAATTCTCCATCCCGACGACAGAAGAGAAAAACGGTCAAAAAGTCGAT GAAGTCTTATTTCGTGGCACG AAACCGATGGAACAACAAATGAAAGACCGTGCAGATGAAGACACTGTTCACGCCTACAAACTTTCCAACCCGACCAAAACCGATACCGGCGTATATGTTGAAGCCATTCACGACATCGGCGATTTCACGCTGACCGGCGGGCTGCGTTACGACCGCTTCAAGGTGAAAACCCATGACGGCAAAACCGTTTCAAGCAGCAACCTTAACCCGAGTTTCGGTGTGATTTGGCAGCCGCACGAACACTGGAGCTTCAGCGCGAGCCACAACTACGCCAGCCGCAGCCCGCGCCTGTATGACGCGCTGCAAACCCACGGTAAACGCGGCATCATCTCGATTGCCGACGGCACAAAAGCCGAACGCGCGCGCAATACCGAAATCGGCTTCAACTACAACGACGGCACGTTTGCCGCAAACGGCAGCTACTTCTGGCAGACCATCAAAGACGCGCTTGCCAATCCGCAAAACCGCCACGACTCTGTCGCCGTCCGTGAAGCCGTCAATGCCGGTTACATCAAAAACCACGGTTACGAATTGGGCGCGTCCTACCGCACCGGCGGCCTGACTGCCAAAGTCGGCGTCAGCCACAGCAAACCGCGCTTTTACGATACGCACAAAGACAAGCTGTTGAGCGCGAATCCTGAATTTGGCGCACAAGTCGGCCGCACTTGGACGGCCTCCCTTGCCTACCGCTTCCAAAATCCGAATCTGGAAATCGGCTGGCGCGGCCGTTATGTTCAAAAAGCTACGGGTTCGATATTGGCGGCAGGTCAAAAAGACCGCAAAGGCAACTTGGAAAACGTTGTACGCAAAGGTTTCGGTGTGAACGATGTCTTCGCCAACTGGAAACCGCTGGGCAAAGACACGCTCAATGTCAATCTTTCGGTTAACAACGTGTTCAACAAGTTCTACTATCCGCACAGCCAACGCTGGACCAATACCCTGCCGGGCGTGGGACGTGATGTACGCTTGGGCGTGAACTACAAGTTCTAAAACGCACATCCCGAAAAAATGCCGTCTGAAAGCCTTTCAGACGGCA

FrpB-mimo amino-acid sequence (start and mimotope insert are in bold andunderlined) MNTPLFRLSLLSLTLAAGFAHAAENNAKVVLDTVTVKGDRQGSKIRTNIVTLQQKDESTATDMRELLKEEPSIDFGGGNGTSQFLTLRGMGQNSVDIKVDNAYSDSQILYHQGRFIVDPALVKVVSVQKGAGSASAGIGATNGAIIAKTVDAQDLLKGLDKNWGVRLNSGFASNEGVSYGASVFGKEGNFDGLFSYNRNDEKDYEAGKGFRNVNGGKTVPYSALDKRSYLAKIGTTFGDDDHRIVLSHMKDQHRGIRTVREEFTVGDKSSRINIDRQAPAYRETTQSNTNLAYTGKNLGFVEKLDANAYVLEKERYSADDSGTGYAGNVKGPNHTRITTRGANFNFDSRLAEQTLLKYGI NYRHQEIKPQAFLNSKFSIPTTEEKNGQKVD EVLFRGTKPMEQQMKDRADEDTVHAYKLSNPTKTDTGVYVEAIHDIGDFTLTGGLRYDRFKVKTHDGKTVSSSNLNPSFGVIWQPHEHWSFSASHNYASRSPRLYDALQTHGKRGIISIADGTKAERARNTEIGFNYNDGTFAANGSYFWQTIKDALANPQNRHDSVAVREAVNAGYIKNHGYELGASYRTGGLTAKVGVSHSKPRFYDTHKDKLLSANPEFGAQVGRTWTASLAYRFQNPNLEIGWRGRYVQKATGSILAAGQKDRKGNLENVVRKGFGVNDVFANWKPLGKDTLNVNLSVNNVFNKFYYPHSQRWTNTLPGVGRDVRLGVNYKF

1. An FrpB protein having one or more deletions of non-conserved aminoacids compared to a corresponding wild-type FrpB protein.
 2. An FrpBprotein in which one or more of the amino acids of at least one of itsloops has been deleted.
 3. The protein according to claim 1 which iscross-protective.
 4. The protein according to claim 1 in which one ormore of the amino acids of at least 2 loops have been deleted.
 5. Theprotein according to claim 1 in which one or more of the amino acids ofloop 7 and/or 5 have been deleted.
 6. The protein according to claim 1in which one or more the amino acids of any one or more of loops 1, 2,3, 4, 6, 8, 9, 10, 11, 12 and 13 have been deleted.
 7. The proteinaccording to claim 1 in which 11 to 33 amino acids have been deletedfrom loop
 7. 8. The protein according to claim 1 in which 23-33 aminoacids have been deleted from loop
 7. 9. The protein according to claim 1in which about 28 amino acids have been deleted from loop
 7. 10. Theprotein according to claim 1 in which 18-29 amino acids have beendeleted from loop
 5. 11. The protein according to claim 1 in which 19-29amino acids have been deleted from loop
 5. 12. The protein according toclaim 1 in which 24 amino acids have been deleted from loop
 5. 13. Theprotein according to claim 1 in which, with reference to FrpB strainH44/76, the amino acid deletion is made in the range of amino acids376-413, or a corresponding deletion made, from loop
 7. 14. The proteinaccording to claim 1 in which, with reference to FrpB strain H44/76, theamino acid deletion is made in the range of amino acids 381-408, or acorresponding deletion made, from loop
 7. 15. The protein according toclaim 1 in which, with reference to FrpB strain H44/76, an amino acidsequence comprising TTEEKNGQKVDKPMEQQMKDRADEDTVH has been deleted, or acorresponding deletion made, from loop
 7. 16. The protein according toclaim 1 in which, with reference to FrpB strain H44/76, the amino aciddeletion is made in the range of amino acids 247-280, or a correspondingdeletion made, from loop
 5. 17. The protein according to claim 1 inwhich, with reference to FrpB strain H44/76, the amino acid deletion ismade in the range of amino acids 252-275, or a corresponding deletionmade, from loop
 5. 18. The protein according to claim 1 in which, withreference to FrpB strain H44/76, an amino acid sequence comprisingQHRGIRTVREEFTVGDKSSRINID has been deleted, or a corresponding deletionmade, from loop
 5. 19. The protein of claim 1 in which the deletedsequence is replaced by another amino acid sequence.
 20. The protein ofclaim 19 in which the sequence is deleted by mutagenesis.
 21. Apolynucleotide encoding the protein of claim
 20. 22. An expressionvector comprising the polynucleotide of claim
 21. 23. A host cellcomprising the expression vector of claim
 22. 24. A method for producingthe protein of claim 1 comprising: culturing the host cell of claim, andrecovering the expressed protein.
 25. A method for refolding an FrpBprotein comprising contacting the FrpB protein with an alkalinerefolding buffer comprising 3-dimethyldodecylammoniopropanesulfonate(Zwittergent 3-12 or SB-12).
 26. A method according to claim 25 whereinthe protein is a protein according to claim
 1. 27. A method according toclaim 25 wherein the refolding buffer comprises ethanolamine and SB-12.28. A method according to claim 27 wherein the ethanolamine is about 20mM ethanolamine.
 29. A method according to claim 25 wherein therefolding buffer has pH11.
 30. A method according to claim 25 whereinthe SB-12 is 0.2% SB-12.
 31. A method according to claim 25 wherein theSB-12 is 0.5% SB-12.
 32. A method according to claim 25 wherein therefolding buffer further comprises guanidium chloride, NaCl and/or urea.33. A method according to claim 32 wherein the refolding buffer furthercomprises guanidium chloride.
 34. A method according to claim 33 whereinthe refolding buffer further comprises 0.4M guanidium chloride.
 35. Amethod of claim 25 comprising the following steps: a. optionallyexpressing an FrpB protein in a host cell; optionally breaking the hostcell to obtain an inclusion body comprising the FrpB protein; optionallywashing the inclusion body; b. optionally solubilisation of at leastpart of the inclusion body and the FrpB protein (preferably withGuanidinium hydrochloride); c. contacting the solubilised FrpB proteinwith the refolding buffer; and d. optionally removing (or changing) therefolding buffer from the FrpB protein.
 36. A refolding buffercomprising ethanolamine, SB-12 and, optionally, guandinium chloride foruse in the method of claim
 25. 37. An isolated, refolded FrpB proteinobtained or obtainable by the method of claim
 25. 38. A pharmaceuticalcomposition comprising at least one FrpB protein of claim 1 or 37, and apharmaceutically acceptable carrier.
 39. A pharmaceutical compositionaccording to claim 38 wherein at least 30%, 50%, 70%, or 90% of the FrpBprotein present in the composition is refolded.
 40. A pharmaceuticalcomposition according to claim 38 in the form of a vaccine.
 41. Thepharmaceutical composition of claim 38 comprising a FrpB protein derivedfrom Neisseria meningitidis.
 42. The pharmaceutical composition of claim38 comprising a FrpB protein derived from Neisseria gonorrhoeae.
 43. Thepharmaceutical composition according to claim 38 wherein saidcomposition comprises at least one other Neisserial antigen.
 44. Thepharmaceutical composition of claim 43 comprising at least one otherNeisserial antigen derived from Neisseria gonorrhoeae.
 45. Thepharmaceutical composition of claim 43 comprising at least one otherNeisserial antigen derived from Neisseria meningitidis.
 46. Thepharmaceutical composition of claim 38 in the form of a subunitcomposition.
 47. The pharmaceutical composition of any one of claim 38in the form of an outer membrane vesicle preparation, or a mixed subunitplus outer membrane vesicle preparation.
 48. A pharmaceuticalcomposition claim 38 further comprising at least one other Neisserialantigen selected from the group consisting of: a. at least oneNeisserial adhesin selected from the group consisting of FhaB, NspA,Hsf, NadA, PilC, Hap, MafA, MafB, Omp26, NMB0315, NMB0995 and NMB1119;b. at least one Neisserial autotransporter selected from the groupconsisting of Hsf, Hap, IgA protease, AspA and NadA; c. at least oneNeisserial toxin selected from the group consisting of FrpA, FrpC,FrpA/C, VapD, NM-ADPRT, and either or both of LPS immunotype L2 and LPSimmunotype L3; d. at least one Neisserial Fe acquisition proteinselected from the group consisting of TbpA high, TbpA low, TbpB high,TbpB low, LbpA, LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB,Bcp, NMB0964 and NMB0293; and e. at least one Neisserial membraneassociated protein, preferably outer membrane protein, selected from thegroup consisting of PldA, NspA, TspA, FhaC, NspA, TbpA(high), TbpA(low),LbpA, HpuB, TdfH, PorB, HimD, HisD, GNA1870, OstA, HlpA, MltA, NMB 1124,NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TspB, PilQ and OMP85. 49.The pharmaceutical composition of claim 38 further comprising one ormore bacterial capsular polysaccharides or oligosaccharides.
 50. Thepharmaceutical composition of claim 49 wherein the one or more capsularpolysaccharides or oligosaccharides is derived from bacteria selectedfrom the group consisting of Neisseria meningitidis serogroup A, C, Y,and/or W-135, Haemophilus influenzae b, Streptococcus pneumoniae, GroupA Streptococci, Group B Streptococci, Staphylococcus aureus andStaphylococcus epidermidis, and are preferably conjugated to a source otT-helper epitopes.
 51. Use of an FrpB protein of claim 1 or claim 37 inthe preparation of a medicament for use in generating an immune responsein an animal.
 52. Use of an FrpB protein of claim 1 or claim 37 in thepreparation of a medicament for treatment or prevention of Neisserialinfection.
 53. A method of preventing or treating Neisserial infectionby administering an FrpB protein of claim 1 or claim 37 to a patient inneed thereof.
 54. The method of claim 52 in which Neisseria meningitidisinfection is prevented or treated.
 55. The method of claim 52 in whichNeisseria gonorrhoeae infection is prevented or treated.
 56. An antibodyimmunospecific for the FrpB protein as claimed in claim 1 or claim 37.57. A pharmaceutical composition useful in treating humans with aNeisserial disease comprising at least one antibody according to claim56 and a suitable pharmaceutical carrier.
 58. Use of the antibody ofclaim 56 in the manufacture of a medicament for the treatment orprevention of Neisserial disease.
 59. The use of claim 58 whichNeisseria meningitidis infection is prevented or treated.
 60. The use ofclaim 58 in which Neisseria gonorrhoeae infection is prevented ortreated.
 61. A method of diagnosing a Neisserial infection, comprisingthe steps of identifying an FrpB protein, or an antibody thereto, withina biological sample from an animal suspected of having such an infectionusing a FrpB protein as claimed in claim 1 or claim 37, or an antibodyas claimed in claim
 56. 62. The method of claim 61 in which Neisseriameningitidis infection is diagnosed.
 63. The method of claim 61 in whichNeisseria gonorrhoeae infection is diagnosed.